Effect of a multiple micronutrient enriched maize-based liquid meal supplement on iron

status of grade 3 and 4 learners attending Sunnyside primary school, Pretoria

by

Lusanda Susan Simelane

Submitted in partial fulfillment of the requirements for the degree

MSc Nutrition

in the Faculty of Natural & Agricultural Sciences

University of Pretoria

Pretoria

Supervisor: Dr Zelda White

Co-Supervisor: Prof Andrea Oelofse

February 2015

DECLARATION

I declare that the dissertation, which I hereby submit for the degree MSc nutrition at the

University of Pretoria, is my own work and has not previously been submitted by me for a

degree at this or any other tertiary institution.

SIGNATURE:……………………………………………… Lusanda Susan Simelane

DATE: February 2015

i

ABSTRACT

Background: About one half of school-age children in developing countries are estimated to

be affected by iron deficiency. Failure to treat micronutrient deficiencies can negatively affect

health and economic development.

Objective: To determine the effect of multiple micronutrient maize-based liquid meal

supplement on the iron status and the nutritional status of primary school children.

Design: A randomized double-blind placebo controlled trial was conducted.

Setting and subjects: The study took place at Sunnyside Primary School in Sunnyside, Pretoria,

in the urban area of Tshwane (Gauteng Province, South Africa). Grades 3 and 4 male and female

learners aged 8-12 years, enrolled in the 2010 academic year were recruited for the study.

Methods: Participants were dewormed, to eliminate parasitic infestaion at the beginning of the

study. The experimental product was a maize-based liquid meal supplement enriched with

macronutrients and micronutrients including chelated ferrous bisglycinate, while the control

had the same macronutrient profile but no added micronutrients. The learners took the meal

supplement every morning on school days for 14 weeks. Iron status was measured by

Haemoglobin (Hb) levels and the nutritional status was measured by anthropometric measures

at baseline and end. Groups were compared with respect to change in Hb and change in

anthropometry using an analysis of covariance (ANCOVA) with baseline Hb values as covariate.

Testing was done at the 0.05 level of significance.

Results: There was no significant difference in the Hb levels at baseline (12.6 ± 1.1 g/dL and

12.8 ± 1.1 g/dL) (P = 0.250) between the experimental and control groups respectively. The

prevalence of mild anemia (Hb< 11g/dL) was low in both the experimental and control groups.

Over the 14 weeks study period, consumption of experimental products was similar and there

was no significant effect on Hb levels of the participants observed (P = 0.806) in the

experimental and control groups. There was also no significant change observed in the

anthropometry of the participants.

Conclusion: The maize-based liquid meal supplement enriched with multiple micronutrients did

not have a significant effect on the iron status of the participants in this study, possibly owing

to low prevalence of anemia, a low rate of consumption and therefore iron absorption.

ii

ACKNOWLEDGEMENTS

I would like to thank the following institutions and persons:

My supervisors, Dr Zelda White and Prof Andrea Oelofse, for their guidance, advice, positive

criticism, patience and support during the course of this study

Prof Piet Becker, for assisting me with the statistical analysis for this study

Resaf Company (SA) for supplying the multiple micronutrient enriched maize –based liquid

meal supplement and supporting the study.

The University of Pretoria for the financial support rendered during my period of study at the

University.

The head teacher and teaching staff of Sunnyside Primary School (Pretoria)

The Grade 3 and 4 learners for the year 2010 of Sunnyside Primary School

Mr Jonathan Kotze for the help rendered during the collection of data.

Dietetics students from University of Pretoria (2010), for the assistance they offered during the

collection of data.

My colleagues, for their support and encouragement

My family and friends, for their continued support, understanding and encouragement

Above all, honour and glory goes to the Lord God Almighty for every good thing comes from

Him.

iii

TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………………………………………………………..…i

ACKNOWLEDGEMENTS……………………………………………………………………………………………………........…ii

TABLE OF CONTENTS………………………………………………………………………………………..…………………….…iii

LIST OF TABLES……………………………………………………………………………………………………………..………..….v

LIST OF FIGURES………………………………………………………………………………………………………………………...v

ABBREVIATIONS………………………………………………………………………………………………………………….…....vi

CHAPTER 1: INTRODUCTION………………………………………………………………………………………………………1

1.1 BACKGROUND ........................................................................................................................... 1

1.2 RESEARCH HYPOTHESIS………………………………………………………………………………………………………..4

1.3 OBJECTIVES ............................................................................................................................... 4

1.4 CONCEPTUALIZATION ............................................................................................................... 4

1.5 STRUCTURE OF THIS DISSERTATION ......................................................................................... 7

CHAPTER 2: LITERATURE REVIEW ................................................................................................... 8

2.1 INTRODUCTION ......................................................................................................................... 8

2.1.1 Focus of literature review…………………………………………………………………………………….……………8

2.2 THE ROLE OF IRON IN GROWTH AND DEVELOPMENT ............................................................. 8

2.2.1 Iron metabolism and homeostasis ........................................................................................ 9

2.2.2 Etiology of anemia ............................................................................................................... 11

2.2.3 Diagnosis of iron deficiency and anemia ............................................................................. 16

2.2.4 Consequences of iron deficiency and anemia ..................................................................... 21

2.2.5. Epidemiology of iron deficiency and anemia...................................................................... 23

2.3 STRATEGIES TO ADDRESS IRON DEFICIENCY AND ANEMIA……………………………….…………….….24

2.3.1 Food fortification ................................................................................................................. 25

2.3.2 Education combined with dietary diversification ................................................................ 28

2.3.3 Supplementation ................................................................................................................. 28

2.4. LITERATURE REVIEW SUMMARY ........................................................................................... 38

CHAPTER 3: METHODOLOGY ........................................................................................................ 39

3.1 ETHICAL CONSIDERATIONS ..................................................................................................... 39

iv

3.2 RESEARCH DESIGN……………………………………………………………………………………………………………..39

3.3 STUDY SETTING ....................................................................................................................... 39

3.4 STUDY POPULATION ............................................................................................................... 39

3.4.1. Recruitment and screening ................................................................................................. 39

3.5 SAMPLING METHOD ............................................................................................................... 41

3.5.1 Sample size ........................................................................................................................... 41

3.6 INTERVENTION…………………………………………………………………………………………………………………..41

3.6.1 De-worming ......................................................................................................................... 41

3.6.2 Experimental product .......................................................................................................... 41

3.7 RANDOMISATION AND BLINDING .......................................................................................... 44

3.7.1 Preparing and administering of experimental product ....................................................... 44

3.7.2 Subject compliance and monitoring .................................................................................... 45

3.7.3 Packaging and blinding of experimental products .............................................................. 45

3.8 DATA COLLECTION……………………………………………………………………………………………………………..45

3.8.1 Screening .............................................................................................................................. 45

3.8.2 Schedule of measurements ................................................................................................. 45

3.8.3 Variables............................................................................................................................... 46

3.9 STATISTICAL ANALYSIS ........................................................................................................... 47

CHAPTER 4: RESULTS AND DISCUSSION ....................................................................................... 48

4.1 CHARACTERISTICS ................................................................................................................... 48

4.1.1 Socio-demographic data ...................................................................................................... 48

4.1.2. Anthropometric characteristics .......................................................................................... 50

4.1.3 Consumption ........................................................................................................................ 52

4.1.4 Iron status ........................................................................................................................... 53

4.2 LIMITATIONS:…………………………………………………………………………………………………………………..57

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS………………………………..………………………….58

REFERENCES .................................................................................................................................. 59

ADDENDUM 1: ETHICAL APPROVAL ........................................................................................... 744

ADDENDUM 2: ASSENT FORM FOR 7-8 YEARS FOR CLINICAL TRIAL/INTERVENTION RESEARCH

v

..................................................................................................................................................... 755

ADDENDUM 3: NAME BADGES…………………………………………………………………………………………………77

ADDENDUM 4: COMPLIANCE SHEETS……………………………………………………………………………………...78

ADDENDUM 5: SOCIO-DEMOGRAPHIC QUESTIONNAIRE………………………………………………………...79

ADDENDUM 6: SICKNESS DIARY……………………………………………………………………………………………….82

LIST OF TABLES

Table 1.1: Conceptual definitions and operationalization .......................................................... 6

Table 2.1: Influence of the iron status on various indicators in absence of other diseases ..... 17

Table 2.2: Stages of anemia and values used in demographic and health surveys .................. 18

Table 2.3: Cut off values for anemia at sea level and above sea level using hemoglobin

concentration ............................................................................................................................ 18

Table 2.4: Adjustments to haemoglobin cutoffs and individual values for altitude and ethnicity

................................................................................................................................................... 19

Table 2.5: Suggested iron fortification compounds for different food vehicles ....................... 26

Table2.6: Overview of iron supplementation trials on iron status of school age children ....... 30

Table2.7: Overview of multiple micronutrient supplementation trial characteristics trilals on

mean Hb concentration in school age children ........................................................................ 34

Table2.8: Nutritional content of the supplements/fortified foods used in multiple

micronutrient studies ................................................................................................................ 36

Table 3.1: Nutritional composition of experimental and control product ............................... 43

Table 4.1: Charactristics of learners who participated in the 14 week study ........................... 48

Table 4.2: The socio-demographic characteristics of the study population ............................. 49

Table 4.3: Anthropometric characteristics of the study population ......................................... 51

Table 4.4: Anemia prevalence in the experimental and control groups at baseline and at

end……………………………………………………………………………………………………………………………………….54

Table 4.5: Iron status of participants at baseline, end and change from baseline to end………54

LISTOF FIGURES

Figure 1: Conceptual framework ................................................................................................. 5

Figure 2: Regulation of intestinal iron uptake ........................................................................... 10

vi

Figure 3: Trial profile of the 14 week intervention study ........................................................ 40

ABREVIATIONS

ADA American Dietetic Association

AGP alpha-1 glycoprotein

AIDS Acquired Immunodeficiency Syndrome

ALA Eicosapentaenoic acid

ALC Active learning capacity

ANCOVA Analysis of Covariance

BAZ Body Mass Index for age z-score

BMI Body Mass Index

CI Confidence Interval

CRP C- reactive protein

DCYTB duodenal cytochrome

DHA Docosahexaenoic acid

DMT1 Divalent metal ion transporter 1

DOH Department of Health

FAO Food and Agriculture Organization

FBDG Food based dietary guidelines

Fe2+ Ferrous ion

Fe3+ Ferric reductase

H+ Hydrogen

HAZ Height for age z- score

Hb Haemoglobin

HCP1 Heme carrier protein 1

HIV Human Immuno-deficiency Virus

ICSH International Committee for Standardization

IDA Iron deficiency anaemia

MVC Mean corpuscular volume

n number

vii

Na+ Sodium

NaFe EDTA Sodium Ferredetate ethylenediamine tetracetic acid

NFCS National Food Consumption Survey

NSNP National Schools Nutrition Program

PSNP Primary School Nutrition Program

RDA Recommended dietary allowance

RDI Recommended dietary intake

SANHNES South African National Health and Nutrition Examination Survey

sd standard deviation

sTfR soluble transferrin receptor

UNICEF United Nations Children’s Fund

UNU United Nations University

WMD Weight mean difference

WHO World Health Organization

WHZ Weight for height z- score

ZnPP Zinc protoporphyrin

1

CHAPTER 1: INTRODUCTION

1.1 BACKGROUND

Micronutrient malnutrition is considered as a public health problem affecting more than 2

billion people worldwide.1 In developing countries the magnitude is much greater because

malnutrition, infection and poverty are most common, and often interlinked.1,2 Failure to treat

micronutrient deficiencies can negatively affect health and economic development.1 Iron,

vitamin A and iodine deficiencies are the major micronutrient deficiencies affecting children

including school children in developing countries. In addition deficiencies of vitamin C, zinc and

B vitamins often occur concurrently with the 3 major micronutrient deficiencies. About one half

of school age children in developing countries are estimated to be affected by iron deficiency. 3

The school age years are therefore an opportune time for addressing iron deficiency because of

the following reasons: iron deficiency impairs fitness and work capacity thus interventions to

improve iron status may enhance fitness and work capacity of children.4 Improving iron status

may enhance learning potential of children.5 Improving iron status of girls may help prevent

anemia in their reproductive years. Most importantly, the school offers an ideal distribution

system for several types of public health interventions.6 Micronutrient deficiencies are also a

risk factor for frequent and severe infections. These infections in turn may have adverse effects

on nutritional status. 7

School children are significantly disadvantaged in terms of nutrition interventions and/or

programs and in urgent need of additional attention, if they are to reach their full

developmental potential.8 The full genetic potential of the child for physical growth and mental

development may be compromised due to deficiency (even subclinical) of micronutrients.

Children and adolescents with poor nutritional status are exposed to alterations of physical,

mental and behavioral functions that can be corrected to certain extend by dietary measures.9

Therefore, in trying to alleviate micronutrient malnutrition, the South African Government

designed a 3-way food-based approach which includes mandatory food fortification. The other

2

two approaches include a micronutrient supplementation program for women and children,

and an educational program to promote better dietary habits, including breast-feeding

initiatives, school feeding program and campaigns to encourage people to grow their own

vegetables and fruits to improve household food security as well as increasing intakes of

micronutrient-rich foods. The approaches are known as the Integrated Nutrition Program

(INP).10

School feeding has the potential to contribute toward alleviating both short-term hunger and

hidden hunger (micronutrient deficiencies) for school children to reach their full mental and

physical potential and perform optimally in school.8 Therefore, to ensure good nutritional status

and improvement of the general health as well as learning capacity, a comprehensive Primary

School Nutrition Program (PSNP) was introduced twenty years back in South Africa. In its first

ten years of implementation PSNP was coordinated by the Department of Health, in 2004 it was

relocated to the Department of Education. The decision was based on consideration that school

feeding had important educational outcomes which are the functional responsibility of the

Department of Education.11It was then renamed the National Schools Nutrition Program

(NSNP). The primary aim of the program was to improve the educational experience of the

disadvantaged primary school learners through promoting punctuality, alleviating short term

hunger, improving concentration and contributing to general health development.12

In 1996, an evaluation on the PSNP showed a high prevalence of malnutrition especially

amongst the black and colored primary school children, and reports have shown poor and

inconsistent coverage of the program in several parts of the country. Numerous challenges

were encountered in the program, such as inappropriate feeding times and food of a sub-

standard quality and quantity.13 Furthermore, several schools were found to have poor

infrastructure to be able to adequately support the implementation of NSNP effectively. Lack of

proper kitchen infrastructure, cooking equipment and storage facilities such as refrigerators for

storing perishables, have been a drawback in the preparation of school meals. As a result, the

complete advantage of school feeding has not been realized. Therefore, micronutrient

3

deficiencies (including iron deficiency) are still highly prevalent in South African school children

despite the existence of a national school-feeding program. School-feeding program often focus

on relieving short-term hunger, and do not always concentrate on alleviating or preventing

hidden hunger.8

To alleviate short term and hidden hunger, deworming, nutrition education and micronutrient

supplementation are recognized as more cost-effective interventions. However, there has been

a lack of systematic implementation of these interventions as part of the NSNP.13 Controversy

surrounding the use of supplementation products as part of NSNP has been another issue. The

concern has been that: use of commercial supplements may defeat the aim of nutrition

education and may not be in line with local eating habits in addition, enriched commercial

foods tend to be more expensive and do not contribute to “community involvement”.

Unfortunately, the chance of local food based meal to provide the same micronutrient

contribution as a meal that includes a fortified product is very slim, unless it contains fortified

ingredients. In addition, food sources of iron are relatively expensive. Alternative sources of

iron which are cheaper include spinach and legumes, but unfortunately their iron content is

smaller and less bioavailable, presenting yet another challenge.13

This study therefore sought to provide solid scientific evidence describing the magnitude of

impact to be expected from enriched maize-based meal supplements on iron status in primary

school children. The results from this project are meant to assist in making informed choices

about the importance and potential impact of multiple micronutrient interventions in primary

school children. The study sought to provide input for the Integrated Nutrition Program of

South Africa, especially the NSNP on the use of maize-based supplementation in school feeding

schemes to reach the most vulnerable groups. The study investigated the effect of a ready to

use multiple micronutrient enriched maize-based liquid meal supplement on iron status of

primary school children aged 8 to 12 years.

4

1.2 RESEARCH HYPOTHESIS

1. Consumption of a multiple micronutrient enriched maize-based liquid meal supplement 5

days a week for 14 weeks, will improve iron status as measured by hemoglobin, in primary

school children (8 – 12 years old). Iron therapy is expected to increase Hb values.14 The

supplement contains Ferrous bisglycinate chelate which has a higher bioavailability (3.4 -4

times higher) than ferrous sulpphate.15

2. Consumption of a multiple micronutrient enriched maize-based liquid meal supplement, will

improve nutritional status. Multiple micronutrient interventions containing iron improve

nutritional status compared to placebo or single nutrient interventions.16

1.3 OBJECTIVES

1. To determine the effect of multiple micronutrient enriched maize - based liquid meal

supplement on the iron status of primary school children using hemoglobin as a biomarker.

2. To determine the effect of multiple micronutrient enriched, maize-based liquid meal

supplement on the nutritional status of primary school children using anthropometry.

1.4 CONCEPTUALIZATION

The conceptual framework used in this study shows that nutrition status results from several

interrelated causes. Anemia, which is one indication of poor nutrition status, is an outcome of

poor diet, increased iron demand, infection and sometimes inherited conditions. Anemia can be

assessed by measuring hemoglobin concentration, and can be classified as mild, moderate or

severe. To correct anemia, a multiple micronutrient dietary supplement which contains

chelated iron can be useful, as this type of iron is more bioavailable, and the presence of the

other nutrients can work synergistically with the iron to correct anemia. If anemia is not

corrected it may result to poor health, low activity, defects in growth, affect cognition, in severe

cases it may lead to death (Figure 1.1)

5

iron supplementation

Figure 1. Conceptual framework17

Inadequate absorption: iron

absorption inhibitors

Inadequate ingestion: iron

deficient meals

Inadequate utilisation: lack of other erythropoietin nutrients

Increased destruction resulting in decreased release from stores: infections

Increased blood loss or excretion: helminth infestation

Increased requirement e.g. physical growth

Consequences: growth abnormalities inadequate muscle function fatigue low cognition increased morbidity

Stages of anemia: Mild:Hb;9,5 – 10.99 g/dL Moderate: Hb;7 – 9.4 g/dL severe: Hb < 7g/dL

Nutrition Management: food diversification food fortification with iron food fortification with multiple micronutrients dietary supplements containing multiple micronutrients

Medical management: iron supplementation multiple micronutrient supplementation

Anemia

6

Table 1.1: Conceptual definitions and operationalization

Terminology Conceptual definition Operationalization

Anemia Condition indicating a deficiency

of the size or number of red

blood cells or the amount of

haemoglobin they contain.17

Indicated by decrease in the

quantity of hemoglobin i.e. <

7g/dL indicate severe anemia,

7 – 9.4 g/dL indicate moderate

anemia and 9.5 – 10.99 g/dL

indicate mild anemia18

Grade 3 and 4 learners Primary school pupils in Grade 3

and 4 classes.

Sunnyside Primary school boys

and girls (8 -12years old)

Multiple micronutrient

enriched maize-based meal

supplement

A 250ml ready to drink, made

from maize meal and enriched

with multiple nutrients including

chelated iron.

Experimental product

Chelated iron Two molecules of amino acid

bound with a covalent bond to

an iron molecule.19

Ferrous bisglycinate chelate

(Ferrochel®)

Hb: Hemoglobin A conjugated protein containing

four heme groups and globin; it

is the oxygen carrying pigment of

erythrocytes. 17

Cut off points indicate iron

status

Biomarker for Iron status

Iron status Can range from overload to

deficiency and anemia

Iron status has a variety of

indicators. Haemoglobin was

used in this study.

Mild anemia Low Hb, but not severe. Hb: (9.5g/dL to ≤10.99)18

7

1.5 STRUCTURE OF THIS DISSERTATION

A chapter format has been used in presentation of this dissertation.

Chapter 1 is an introductory chapter, followed by Chapter 2, which is a review of the literature.

This review covers the nutrition status of school children including their iron status, the role of

iron in growth and development, iron metabolism, the epidemiology of iron deficiency anemia.

The etiology of iron deficiency anemia (IDA) and the involvement of other micronutrients in the

cause of IDA are also dealt with in this review. Also forming part of the review are the

consequences of IDA, methods of diagnosis as well as the strategies for fighting anemia. A

Review on randomized controlled trial studies, on dietary supplements involving primary school

children are also laid out in Chapter 2.

Chapter 3 shows Methodology including: ethical approval of the study, description of the study

design, the recruitment of the subjects, inclusion and exclusion criteria, screening,

randomisation, blinding, anthropometric and hemoglobin assessment methods as well as the

data collection, capturing and analysis methods used in this study.

Presentation of the results and its discussion is in Chapter 4. In this chapter the description

(demographic information) of the participants is presented. Baseline and end assessment data

are reported in this chapter. In the discussion the results are compared to available literature

and possible interpretation for results is given. Limitations of this study are reviewed in this

chapter.

Chapter 5 gives a conclusion based on all assessments. Recommendations for future research

are given in this chapter.

8

CHAPTER 2: LITERATURE REVIEW

2.1 INTRODUCTION

School age children suffer from multiple micronutrient deficiencies like most people in

developing countries.20 It is estimated that 13 – 27% pre – school children have two or more

micronutrient deficiencies, indicating that 100millions of these children are affected.21 This

indicates that if these children’s condition is not corrected they will move on to primary school

with the same or even worse nutritional status. Reports of impairment in growth, immune

function and cognitive performance have been made, concerning school age children who are

deficient in iron, zinc, vitamin A and iodine.22 Reduction in both productivity and cognitive

performance in adult hood can occur due to the health consequences of micronutrient

deficiencies. Therefore, reducing the prevalence of micronutrient deficiencies is of importance

to several policy makers in developing countries.23

Malnutrition has a negative impact on morbidity, mortality, educability and productivity. In

South Africa, the nutritional status of the population has not improved over the last fourteen

years except for the folate and iodine status. The prevalence of micronutrient deficiencies (i.e.

vitamin A and iron) has increased the double burden of disease in the population.24

Micronutrient deficiencies usually occur concurrently, they tend to interact and coexist. For

example, iron deficiency and vitamin A deficiency usually occur concurrently in the same group

of people. Thus, providing vitamin A supplements for example, can have a positive outcome on

vitamin A status and can improve iron metabolism in affected groups.20

2.1.1 Focus of literature review

The literature review focuses on the role of iron in growth and development, iron metabolism

and homeostasis, etiology of iron deficiency anaemia, diagnosis of iron deficiency anaemia,

epidemiology of iron deficiency and anaemia, iron status and strategies to address iron

deficiency and anaemia.

2.2 THE ROLE OF IRON IN GROWTH AND DEVELOPMENT

Iron is a component of every living cell primarily involved in transport and storage of oxygen,

9

oxidative metabolism and several physiological processes. It is necessary for cellular growth and

functioning.25 Together with other micronutrients, iron is necessary for promotion of physical

growth, sexual maturity and neuromotor development. A number of vitamins and trace

minerals including iron play an important role in boosting both cell-mediated and humoral

immune body defenses. Production of various enzymes, hormones and biochemical mediators

for controlling biological processes and energy production, are shared function of iron with

vitamins and other trace minerals.26 Iron plays a very crucial role in the functioning of the

neurotransmission system through production of dopamine and serotonin.26 The content of

iron in the brain is lowest at birth and increases with age, and reaches adult concentration after

puberty.27 Iron requirements are most likely to exceed intake at 6 – 8months after birth and

during adolescence (for girls).25

2.2.1 Iron metabolism and homeostasis

Iron is a main component of hemoglobin (Hb), needed for basic cellular function in all human

tissues, especially the muscles, brain and blood cells.28 Human beings cannot actively excrete

iron, therefore iron concentration is controlled in the proximal small intestine, at the site of iron

absorption (Figure 2).29 The haem and non haem iron from the diet have specific transporters.

Iron deficiency and hypoxia up regulate heme carrier protein1 (HCP1), a putative haem

transporter.30,31 The divalent metal ion transporter 1 (DMT1), mediates the transport of non

haem iron from the intestinal lumen to the enterocytes.32 DMT1 can only transport ferrous iron

and yet most of the iron enters the duodenum in a ferric form. It is therefore necessary that it

must first be reduced to ferrous iron, ferric reductase, duodenal cytochrome b (DCYTB),33 or

possibly by other reducing agents, such as vitamin C.

10

Figure 2. Regulation of intestinal iron uptake29

HCP: Heme carrier protein; DCYTB: duodenal cytochrome b; DMT1: The divalent metal ion

transporter 1.

Fe2+: ferrous iron: Fe3+: ferric reductase: H+: Hydrogen: Na+: Sodium

Iron that is not transferred to the circulation is stored as ferritin and, is finally lost when the cell

sloughs off at the villus tip. Movement of iron across the basolateral membrane is controlled by

ferroportin 1 and the iron oxidase, hephaestin. The transport protein ferroportin also mediates

iron movement form other cells, including macrophages. Hypoxia and iron deficiency

encourages DMT1, DCYTB and ferroportin stimulation thus increasing iron uptake, iron

deficiency increases iron uptake.33

Hepcidin, a hormone produced by the liver inhibits both absorption and release of iron from

macrophages and other cell types. Therefore, during iron deficiency hepcidin secretion from

the liver is decreased to enable maximum iron absorption.34,35 In the erythroid iron cycle, old

red cells are broken down in the spleen by macrophages, secreted iron returns to the

circulation and binds to transferrin receptors (TfRs) in the bone marrow on erythroid

precursors, and completion of the cycle occurs on entrance of the erythrocytes into the

circulation in the 7-10 days that follows. Iron deficiency encourages maximum iron transfer

through the cycle by increasing expression of ferroportin on macrophages hepatic synthesis,29

11

and TfR1 expression in the bone marrow and other tissues.36 Although the body’s homeostatic

mechanisms are efficient in iron conservation, deficiency of iron can still occur, particularly

when physiologic needs exceed intake or in the case of depletion of iron stores.14

2.2.2 Etiology of anemia

A number of factors may contribute to the development of iron deficiency; low intake and poor

absorption of iron from the diet due to the presence of iron absorption inhibitors such as

polyphenols and phytates, or lack of absorption enhancers such as poor ascorbic acid and meat

intake. High physiological demands of iron during menstruation, pregnancy, and growth may

also contribute to iron deficiency.37,38 Other risk factors include heavy menstrual blood loss,

parasitic infection, acute infection, other micronutrient deficiencies, haemoglobinopathies,50

Human Immune Deficiency Virus (HIV), and other chronic diseases.40,41

2.2.2.1 Iron content in the diet

There are two forms of iron that can be taken up from the diet, haem and non haem iron.

Haem iron is usually obtained from the hemoglobin and myoglobin in animal foods, whereas,

non haem iron comes from cereals, pulses, fruits, and vegetables.42 A majority of diets in

developing countries comprise mainly of cereals and pulses, which means that the form of iron

such a population is likely to get from its diet is non haem iron. Non haem iron, however, has a

low bioavailability.43

2.2.2.2 Bioavailability of iron

Iron bioavailability is defined as the amount of ingested iron which is absorbed and used for

metabolic functions.43 Meals can be categorized into three broad categories in terms of their

iron bioavailability; low, intermediate and high bioavailability.42 The low bioavailability diet

consist of cereals, roots and/tubers and a negligible amount of meat, fish or ascorbic acid. Such

meals have absorption of approximately 5%. Intermediate bioavailability diets have absorption

of approximately 10% and usually consist of cereals, roots and/tubers and negligible food of

animal origin and/vitamin C. The high bioavailability diet is usually composed of generous

quantities of meat, poultry fish, and/foods containing high quantities of ascorbic acid. The iron

absorption of such meals is approximately 15%. The regulation of iron absorption is usually

12

regulated by the iron status; people with normal or high iron stores have a low iron

absorption.46 Sustained negative iron balance can lead to anemia.44 Excessive iron absorption

can have negative effects on the body; it can cause diseases such as hepatic cirrhosis and

diabetes mellitus.47

2.2.2.3 Parasitic infestation

Approximately 35% (320 million) of school age children have round worm infestation; 25% (233

million) are infested with whipworm, and 26% (239 million) have hookworm infestation.48,49,50

Intestinal worms alone account for 11 % and 12% of the total disease burden in 4 -14 years old

boys and girls (from low income countries) respectively.51 It is possible for children to be

concurrently affected by a number of parasitic species.52 Worm infestation may build up over

time and may cause chronic and long lasting health problems. Worms can contribute to

malnutrition through causing lack of appetite, malabsorption and anemia may occur due to

blood loss.52 Parasitic infestation is the most significant infection causing blood loss at the site

of feeding, it also exacerbates bleeding by the secretion of anticoagulants and interferes with

iron uptake in the duodenum as it impairs appetite (with moderate to heavy infection).41 A

relationship between infection intensity and hemoglobin levels has been shown in several

studies and increase in worm infestation results in decreased hemoglobin levels.53,54,55

A study done in East Africa showed that the degree of iron deficiency anemia due to hook

worm infestation was dependant on the intensity and duration of the infection, the iron stores

of the host as well as the species of hookworm. Therefore, Ancylostoma duodenale was more

significant in the prevalence of IDA compared to Necator Americanus.56

Malaria is another parasitic infection which occurs mostly in tropical and sub tropical countries.

Malaria can contribute to iron deficiency and anemia.24 In a cross sectional study from Nigeria,

school children were evaluated to determine the effect of low level Plasmodial infection. The

results indicated that even low level plasmodial infection contributes to anemia.57

2.2.2.4 Other micronutrients in the etiology of anemia

Populations in developing countries are usually affected by concurrent micronutrient

13

deficiencies.29 African school children are vulnerable to coexisting deficiencies of vitamin A and

iron.58,59 A survey carried out in Sri Lanka adolescents reported that 54% males and 55%

females had folate and zinc deficiencies. Moreover, 30% males and 48% females were iron

deficient. The odds ratio of having at least two deficiencies at a time among the iron deficient

children were; 1.6 (95%CI: 0.6 - 4.2) in boys and 0.8 (95%CI: 0.5 - 1.5) in girls. One micronutrient

deficiency could negatively affect the absorption, metabolism and/excretion of another

micronutrient, hence the coexistence. For example, iodine deficiency goiter may be aggravated

by iron deficiency anemia.59

Other micronutrient deficiencies such as riboflavin, folate, vitamin C, A, and B12 may affect

hemoglobin synthesis either by weakening erythropoiesis or indirectly by affecting uptake or

mobilization of iron. Nutrient deficiencies also weaken immune response against helminthic

infections.60

Vitamin A

Vitamin A deficiency and anemia have long been recognized to be linked. Positive correlation

between serum retinol and hemoglobin concentrations have been reported in surveys carried

out in developing countries. Populations with low vitamin A showed a stronger association

between serum retinol and hemoglobin concentrations.61,62

Vitamin A status has an effect on mobilization of iron stores.29 In a study where children were

given soup fortified with iron and vitamin C, an increase in serum iron levels and transferrin

were reported when serum retinol levels were > 40ug/dL than when they were < 20ug/ dL.63

Hemoglobin increases in response to improved vitamin A status in pre-school and primary

school children.63,64

Anemic school children in Tanzania were given on daily basis a placebo, vitamin A (1.5 mg RE),

iron (40mg), or iron plus vitamin A. An increase in hemoglobin was reported in the following

fashion; 3.6g/L, 13g/L, 17.5g/L, and 22.1g/L in the placebo; vitamin A (1.5 mg RE); iron (40mg),

and the iron plus vitamin A groups respectively.64 Anemic and vitamin A deficient pregnant

14

women in Indonesia received a placebo; vitamin A (2.4 mg RE/d), iron (60mg/d) or iron plus

vitamin A 60 mg iron/d, 2.4 mg RE/d.65 Biochemical measures after 8 weeks showed that 16%,

35%, 68% and 97% of pregnant women respectively were no longer anemic. The suggestion is

that dual fortification or supplementation with iron and vitamin A is more effective in

controlling iron or vitamin A deficiency, compared to individual micronutrient fortification or

supplementation.29

Vitamin A status affects anemia in many ways66as follows: vitamin A deficiency results in

decreased resistance to infection, therefore, it gives rise to anemia of infection. Vitamin A

affects iron absorption and/or metabolism, and it is directly involved in the modulation of

erythropoiesis. Given the high incidence of infectious diseases in developing countries, vitamin

A deficiency may aggravate infection, thereby perpetuating anemia of infection.67

Riboflavin

Low intakes of meat and dairy products increase the risk of riboflavin deficiency. In both

developing and developed countries, school children are highly vulnerable to riboflavin

deficiency68 which may affect erythropoiesis thus contributing to the aetiology of anemia (Hb <

11g/dL).69,70 The mechanism by which this impairment occurs is through reduced mobility of

stored iron,71,72 diminished iron absorption and increased iron losses.73,74

A more effective way of improving iron status in adult males and children is to give riboflavin

and iron supplements together.75,76 Three trials carried out with children as well as pregnant

women compared the efficacy of iron supplementation given alone to iron supplementation

together with riboflavin. It was found that dual supplementation enhanced hemoglobin

production. However, the folic acid given with iron may have compromised the accuracy of the

results obtained with the former.77,78 In another trial, riboflavin and iron supplementation

produced no better results than iron supplementation alone.79 Likewise, a trial with Croatian

children showed no added benefit of riboflavin supplementation in school children with

adequate hemoglobin levels.80 The results of these studies suggest that the effect of riboflavin

on hemoglobin status varies and can be affected by a number of factors.29

15

Zinc

Factors that affect iron bioavailability, such as low meat, high phytate and polyphenol intake,

are the same as those affecting zinc absorption.81 Although the data do not suggest that zinc

deficiency plays a role in anemia, iron and zinc deficiencies often coexist and supplements

containing these elements may therefore prove helpful in vulnerable populations.29 However,

numerous studies have reported reduced iron efficacy when zinc and iron are taken

concurrently, possibly due to impairment of iron absorption. An increased intake of non-heme

lowers the biovailability of zinc.82,83 On the contrary, a high dietary zinc to iron ratio can inhibit

iron absorption.104,105 This effect was demonstrated in a study where adults received

micronutrients in water solution,104,05but not when micronutrients were added as dietary

supplements for infant formulations or maize meal preparations.83,85,86 The exact nature of the

mechanism involved in this instance is not clear, but it is likely to be due to competition for

uptake in the eneterocyte. Both iron and zinc ions depend on DMT1 for transportation.32 It is

therefore possible that high zinc concentrations lower iron uptake by the intestines even

though this effect has not been demonstrated in mammalian systems.29

In a randomised controlled supplementation trial in Vietnam, infants were given a daily dosage

respectively alternated as (placebo, 10 mg iron, 10mg zinc or 10mg iron plus 10mg zinc). It was

reported that the zinc and iron supplements were as effective as the iron supplementation

alone in combating iron deficiency and anemia.87 Similar effects were reported in a study

carried out in Mexican children.88,89 Contrasting results were reported in an Indonesian study

conducted with children who received a similar dose for the same amount of time as the

Vietnamese children.87 In the Indonesian study, iron supplementation alone had a better effect

on iron status than combined iron and zinc supplementation, which suggests that the efficacy

of iron absorption is reduced when zinc and iron supplementation coincide.90 The difference in

results of these two studies is attributable in principle to the baseline iron status of the

Vietnamese children,85 who had severe anemia compared to their Indonesian counterparts.90

According to a review by Fischer Walker et al. 2005,91 iron status is not affected when zinc

supplements are given alone. The same review also suggests, however, that iron status is not

16

improved beyond the effect of iron supplementation on its own when zinc and iron

supplements are given concurrently. More studies are therefore needed to investigate the

interaction between the two nutrients.

Folate and vitamin B12

Macrocytic anemia, a type of anemia where the red cells were found to be larger than normal

was discovered by the end of the nineteenth century. Poor intake of folate from the diet and

insufficient folate absorption and utilisation, contributes to suppression of bone marrow

proliferation as part of macrocytic anemia.92 Vitamin B12 deficiency can also contribute to

macrocytic anemia. This anemia is characterised by abnormal red cell precursors in the bone

marrow called megaloblasts. Iron deficiency anemia can occur concurrently with folate and

vitamin B12 deficiency anemia, which results in normocytic anemia. As a result, it may be

difficult to diagnose iron deficiency anemia.93

2.2.3 Diagnosis of iron deficiency and anemia

Iron deficiency can be diagnosed by using a number of indicators such as clinical indicators

where chronic fatigue is usually important. However, clinical indicators are usually not specific

symptoms.14 Dietary evaluation can also be done to assess how much haem and non haem iron

is taken in the diet. The dietary method might also be helpful but better diagnosis relies on

biochemical indicators, particularly for the early stages of deficiency.94

The three stages of iron deficiency are characterised respectively by depletion of iron stores,

followed by iron deficiency eryhropoiesis and iron deficiency, in that order. The first one

involves the depletion of iron stores, the second one is iron deficiency erythropoiesis, and the

third one is iron deficiency anemia. All these stages can be analysed biochemically (Table 2.1).94

Iron deficiency (usually defined as ferritin level < 12 ug/L) is the most prevalent nutritional

deficiency.15 Iron deficiency anemia occurs when there is severe iron deficiency that causes

reduced erythropoiesis, thus reducing the red blood count, which leads to anemia (Hb level <

11.5g/dL).95

It has been agreed that iron status is best determined with the aid of measurements of

hemoglobin, ferritin, soluble transferrin receptor (sTfR), as well as chronic infections serving as

17

indices. However, this procedure is usually expensive and difficult.94 Hemoglobin therefore has

been successfully used in situations where there were financial constraints and field work in

remote areas.96

Table 2.1: Influence of iron status on various indicators in absence of other diseases94

Hb Ferritin (ug/L) STfR

Iron overload Above cut off > 300 Low

Normal Above cut off 100+/-60 Normal

Depleted iron status Above cut off <20 Normal

Iron deficient

erythropoiesis

Above cut off <12 High

Iron deficiency

anemia

Below cut off <10 High

2.2.3.1 Iron status indicators

A number of factors can affect an individual’s iron status, including limited food choice due to

poverty, micronutrient deficiencies, or interaction between nutrients and helminth

infestation.131 Parasitic infestation can affect iron status due to loss of blood, reduced appetite

and lowered rate of absorption.132

A national food consumption survey conducted in 1999 showed that for South African children

as a whole, the intake of calcium, iron, zinc, selenium, vitamin A, D, C and E, riboflavin, niacin,

vitamin B6 and folic acid were below two-thirds of the Recommended Dietary Allowance.

109Children living in urban areas, however, had a significantly higher iron intake (p < 0.05 –

0.001) than those living in rural areas.109 A more recent food consumption survey found that

the prevalence of poor iron and vitamin A status in children in the country appears to have

increased compared with previous national data. In addition, 45.3% of children nationally were

found to have an inadequate zinc status and to be at risk of zinc deficiency.133

A study by Keskin et al. (2005) showed that the prevalence of iron deficiency was relatively

high among school boys of low socio-economic status (SES). Higher tea intake and lower intake

of citrus fruits, red meat and fish among the low SES group, were cited as the major reason for

18

the results obtained in the study.134Biochemical indicators that can be used as indicators for

iron status, include hemoglobin, ferritin and sTfR. Other parameters include, hematocrIt, iron

saturation of plasma transferrin, and zinc protoporphyrin (ZnPP).94

Hemoglobin (Hb)

Anemia can be diagnosed by administering Hb tests. This is an inexpensive and common

measurement. However, hemoglobin concentration, can be affected by a variety of conditions

and diseases, and in any case only becomes noticeable in the third stage of iron deficiency. It

may therefore be necessary to use very specific and sensitive indices to determine whether iron

deficiency is the specific cause of anemia.94 However, hemoglobin measurement alone can be

used to assess prevalence and etiology of anemia when it is not feasible to use multiple

biochemical tests for iron status due to cost or other operational limitations.96 Anemia is graded

variously as mild, moderate or severe anemia (Table 2.2).97

Table 2.2: Stages of anemia and values used in demographic and health surveys 97

Anemia measured by hemoglobin (g/dL)

Anemia Mild Moderate Severe

Children 6-59 months

<11.0 10-10.9 7.0-9.9 <7.0

Children 5-11 years

<11.5 10-11.4 7.0-9.9 <7.0

Children 12-14 years

<12.0 10-11.9 7.0-9.9 <7.0

Non-pregnant women above 15 years

<12.0 10-11.9 7.0-9.9 <7.0

Men (above 15 years)

<13.0 12-12.9 9.0-11.9 < 9.0

Note: Hemoglobin values change with altitude.

Hemoglobin levels depend on factors such as age, sex, biological variation, race, pregnancy,

altitude (Table2.3), iron deficiency anemia, other micronutrient deficiencies, parasitic infection,

certain disease state as well as cigarette smoking.18 Table 2.4 illustrates the adjustments that

need to be made to Hb cutoffs for altitude and ethnicity.18

19

Table 2.3: Cut-off values for anemia at sea level and above sea level using hemoglobin

concentration 98,99

Target Age Hb at sea level (g/dL)

Hb above sea level > 1.500m (g/dL)

Hb above sea level > 2.700m (g/dL)

Infants a 6 -11 months < 11.0 < 12.0 < 13.0 Children 1 – 4 years < 11.0 < 12.0 < 13.0

School age 5 – 11 year < 11.5 < 12.5 < 13.5 School age 12 – 13 years < 12.0 < 13.0 < 14.0 Pregnant women < 11.0 < 13.0 < 14.0 Non pregnant women

< 12.0 < 12.0 < 13.0

Men < 13.0 < 14.0 <1 5.0

Iron deficiency is rare among infants of an age below six months, unless the birth weight is low.

Hemoglobin is best determined using venous blood anticoagulated with EDTA. Blood from the

heel, ear or finger pricks collected in heparinised capillary tubes can be used as an

alternative.100 A cyanmethemoglobin method is most reliable, provided the blood specimens

are correctly diluted. This method is also recommended by the International Committee for

Standardization in Hematology (ICSH).101

Table 2.4: Adjustments to hemoglobin cutoffs and individual values for altitude and ethnicity18

Adjustment to hemoglobin cut-off value (g/dL)

Altitude (m) > 1250, < 1750 +0.5

Ethnicity: African extraction -1.0

The method involves converting all the encountered form of hemoglobin into

cyanmethemoglobin, which is then analysed with a spectrophotometer.101 Hemoglobin levels

can also be determined from field-collected blood spots.102Alternatively a portable hemoglobin

photometer can be used in remote field settings. The HemoCue is a battery-operated device

that uses a dry reagent (sodium azide) in a microcuvette for direct blood collection and

measurement. The accuracy and precision of hemoglobin values based on the HemoCue are

20

comparable to those obtained by following standardised cyanmethemoglobin-based

procedures and methods.103

Ferritin

Ferritin is currently the most useful indicator of iron status. It is the most sensitive parameter in

detecting the first stage of iron deficiency. Plasma content correlates well with iron stores,

hence a lowered, ferritin concentration may indicate depletion of iron stores. However, ferritin

can also be increased by other factors such as infection and inflammation, which means high

ferritin level may not always be an indication that the iron status is within acceptable limits. To

minimize this problem, therefore, chronic and acute infection parameters must also be

measured to determine whether a raised ferritin level is attributable to infection.94 C-reactive

protein (CRP) is currently used to detect the presence of acute infection, while alpha-1

glycoprotein (AGP) is used for chronic infections. A ferritin value below 10ug/L shows definite

iron deficiency despite unclear cutoff values. Another indicator such as sTfR may be used as it is

not likely to be influenced by infection.94

Soluble transferrin receptor

Iron status can be reliably determined with the aid of sTfR where infection is a factor. Iron

requirement has an effect on the release of sTfR from the cells into the blood stream. In the

second stage of iron deficiency sTfR concentration is increased if the Hb concentration remains

above cutoff level after the iron stores are exhausted. Therefore, sTfR is less sensitive than

ferritin but more sensitive than Hb.94 Bone marrow staining is by far the gold standard in

defining iron deficiency.94

Other iron status indicators

(i) HematocrIt: This parameter usually correlates with hemoglobin, but is relatively insensitive

compared to Hb. It is therefore, not a good diagnostic nutritional anemia indicator94

(ii) Iron saturation of plasma transferrin (ratio of plasma iron to total iron binding capacity) and

mean corpuscular volume (MCV): These two indicators are well established and inexpensive to

measure when hematology analysers are available. Iron deficiency is marked by low saturation

of transferrin with iron and decreased size of erythrocytes. Specificity of these indicators is low,

due to the large number of clinical disorders that may affect transferrin saturation.104 Plasma

21

has a diurnal variation and MVC can therefore only indicate the late stage of iron deficiency. It

may be difficult to take these measurements accurately without analysers, as measurements

may be difficult and likely to have errors.96 Ferritin or sTfR are important alternatives in such

situations.94

(iii) Zinc protoporphyrin (ZnPP)

Iron in protoporphyrin is replaced by zinc in cases of iron deficiency and can be measured with

the aid of haematoflourometry,96at the second stage of iron deficiency before Hb levels decline

below cutoff, thus making ZnPP a more sensitive indicator than Hb. However, note that ZnPP

can be influenced (increased) by lead levels.104

2.2.4 Consequences of anemia

As noted, the final stage of iron deficiency is iron deficiency anemia, which is characterised by

low hemoglobin levels105and has been reliably found to retard physical development,

undermine the immune function, inhibit growth and advance onset of fatigue. Cognitive

function and school achievement can also be affected by iron deficiency anemia.106According to

the World Health Organization (WHO), 8000 000 deaths each year are attributable to iron

deficiency anemia. With regard to loss of healthy life, iron deficiency anemia accounts for 25

million disability-adjusted life years.107

2.2.4.1 Effect of on anthropometry

In reality, the double burden of disease has become more severe with the increased prevalence

of micronutrient deficiencies (vitamin A and iron) together with high levels of overweight and

obesity.24 Adding micronutrients to children’s supplementary feeds and fortification of food

have frequently proved to alleviate micronutrient deficiencies and thus helpful in improving the

population’s well being.109 A study conducted in India showed an increase in the mean height-

for-age z-score (HAZ) among school children after they had taken a multiple micronutrient

fortified drink for 14 months.110

According to a review of studies including infants, pre-school and school children, there is a

positive correlation between iron supplementation and linear growth of anemic children.111 In a

22

study by Chwang et al (1988),112an increase in height, weight, and arm circumference

(compared to a control group) was observed in anemic school children who were given iron

supplements for 12weeks. In a study on anthelmintic treatment and iron fortification

conducted with iron deficient primary school children in South Africa, the height-for-age and

weight-for-height z-scores of the subjects was found to have improved significantly.113

Conversely, however, in some studies conducted with iron-replete children it was found that

iron supplementation had proved counterproductive, while in others a similar group of subjects

had proved unaffected by supplementation.113,115 The inconsistency in the results of these

studies could be due to coinciding multiple deficiencies, variation in the duration of studies and

the iron dosages used, different age groups and different degrees of iron deficiency.116

2.2.4.2 Effect on immunity

In cases of infection occurring in the presence of iron deficiency with or without anemia,

normal resistance mechanisms including functioning of phagocytic, T- and B- cells, may be

compromised while the infection lasts, because large doses of iron given to such children may

aggravate the infection. This is because the infectious organism also gets supplied with the iron

resulting in its replication before the immune system of the host has had time to recover.118

Thus deficient as well as excessively high iron levels could compromise the immune function,

which suggests that an iron status within normal parameters should be sought that would

ensure a complete phagocytic and immune response to pathogens.118

Untargeted supplementation in tropical countries where malaria transmission is high, was

found to be associated with an increased risk of severe infection.119,120 Hence the WHO has

suggested (in light of the potential adverse effects of supplementation on malaria infected

individuals) that, iron and folic acid supplementation should be given to anemic children who

are at risk of iron deficiency, and that in such instances concurrent protection against malaria

(such as treated bed nets and anti-malarial drugs) and other infectious diseases should be in

place.

23

2.2.4.3 Effect on cognition and school performance

Results from several randomised trials have shown a causal relationship between iron

deficiency and deficient cognitive function, also suggesting that short-term iron

supplementation can reverse some aspects of impaired cognition. Children suffering from

anemia have demonstrated poor physical and cognitive development. Anemia results in severe

lethargy and low physical capacity for activity, which negatively affect the time spent by

children playing and exploring.122

A study carried out in Malawi demonstrated a significant increase in fluid intelligence in school

children supplemented with iron for 10 months.123 A study carried out in Thailand reported a

great difference between the scores obtained respectively by anemic iron-deficient and iron-

replete children in a Thai language test, as well as in a test gauging general reasoning ability.

The same difference was not evident in arithmetic scores obtained by the same group,

however.124 On the other hand differences between scores of Indonesian school children who

were iron deficient and iron-replete, respectively, were not markedly different for a number of

exams, as well as a test for concentration. However, scores across the board were improved for

the same exams as well as the concentration test as a result of iron supplementation.125

Amplified vulnerability to infections as a result of iron shortage in school children could lead to

lowered school attendance, which could therefore compromise performance.105 Fewer school

days were missed by children fed with biscuits fortified with multiple micronutrients than by a

control group because the intervention had caused a decline in respiratory and diarrhea related

illness.105 A review by Taras (2005) demonstrated an association between iron deficiency and

poor academic performance.126 However, academic performance (at school) improved as result

of iron supplementation administered to normalize depleted iron stores.126

2.2.5. Epidemiology of iron deficiency and anemia

Iron deficiency (ID) and iron deficiency anemia (IDA) are prevalent in women and young

children. More people in the world are affected by iron deficiency than any other type of

malnutrition.95 It is estimated that more than 2 billion people are affected by iron deficiency,

24

and 1.2 billion of these suffer from iron deficiency anemia.127 Anemia is most prevalent in

developing countries, thus 39% of children < 5 years old, 48% of children 5-14 years old, 42% of

all women and 52% of expectant women are suffering from anemia. About 50% of anemia is

due to iron deficiency.97 Estimates show that 53% or 210 million school age children suffer from

IDA.44,45 A recent South African national health and nutrition examination survey showed that,

provincially, the prevalence of iron depletion was the highest in women from Gauteng (11.2%)

and lowest in Eastern Cape women (0.7%).128 Prevalence among younger South African women

was higher (10.5%) than among older women (8.5%). Reports suggest that Asia has the highest

IDA prevalence (58.4%), followed by Africa (49.8%).129 Several studies have been done to try

and capture the IDA prevalence in school children.

A survey of nearly 14000 rural school children in Africa and Asia, showed that IDA prevalence

was more than 40% among children aged 7-11 years old in five African countries (Mali,

Tanzania, Mozambique, Ghana and Malawi).130 IDA prevalence in Asian children aged (7- 11

years) in Vietnam and Indonesia was low (12 and 28 % respectively). Prevalence was found to

be higher among the older than the younger group. Boys had a higher hemoglobin

concentration than girls. However, the IDA prevalence was higher in boys than in girls.130

Results could have been attributable to a higher incidence of parasitic infections among boys, a

higher growth rate (e.g. onset of a more pronounced “growth spurt” than among girls), or other

confounding factors. Certainly a variety of causes are possible.

2.3 STRATEGIES TO ADDRESS IRON DEFICIENCY AND ANEMIA

There are three main strategies for correcting iron deficiency in populations, and they can be

used alone or in combination.125These strategies are: education combined with dietary

modification or diversification, or both to improve iron intake and bioavailability; iron

supplementation and iron fortification of foods. A new approach is biofortification via plant

breeding or genetic engineering. Dietary modification and diversification are the most

sustainable approaches. However, it may be difficult to change dietary practices and

preferences. Moreover, good sources of highly bioavailable iron are expensive.125

25

2.3.1 Food fortification

Even though iron is the most difficult mineral to add in food and ensure adequate absorption,

iron fortification of foods is still the most practical, sustainable and cost-effective long-term

solution to combating iron deficiency.100,101,102 Fortification of staple foods is even more

important as a long-term strategy for addressing micronutrient deficiencies, including iron

deficiency.43 Different foods can be used as vehicles for several iron fortificants (Table 2.5). In

South Africa the fortification of bread, flour and maize meal was legislated in 2003.108 Maize

and wheat flour are currently fortified to provide a person of 10 years or older with electrolytic

iron (25% from unsifted maize and 50% from maize meal) of the recommended dietary

allowance.108

The most bioavailable iron compounds often lead to the development of unacceptable sensory

changes, such as off flavours and colour change.103 Therefore less soluble forms of iron in low

doses are usually used to avoid organoleptic changes.29 Fortification may be the safest

intervention as low doses similar to the physiological environment are used.101,121 Analysis of

studies where infants received iron fortified foods showed no adverse effects and

demonstrated a significant protection effect against development of respiratory tract

infections.119

Most staple foods contain some iron, however, the quantities differ with the different

cultivars.135 This suggests that selective breeding (biofortification) might increase the iron

content of staple foods.29 But then the high phytate content of most staple foods could still

pose a challenge when it comes to bioavailability. Therefore breeding should also be aimed at

producing cultivars low in iron absorption inhibitors.29 A study in which the aim was to lower

the phytic acid content of rice was done by Lucca et al. (2001), it involved introducing phytase

from Aspergillus fumigates.136 The results indicated a seven fold increase in phytase activity.136

Some studies showed that iron uptake from the soil could be increased by introducing a ferric

reductase gene into the plant root systems.137 Breeding or genetic engineering can be useful in

increasing iron content in staple foods.29

26

Table 2.5: Suggested iron fortification compounds for different food vehicles138

Food vehicle Iron Fortificant

Low extraction (white) wheat flour or degermed corn flour

Dry ferrous sulfate Ferrous fumarate Electrolytic iron (2x amount) Encapsulated ferrous sulfate Encapsulated ferrous fumarate

High extraction wheat flour, corn flour, corn masa flour

NaFeEDTA Ferrous fumarate (2x amount) Encapsulated ferrous sulfate (2x amount) Encapsulated ferrous fumarate (2x amount)

Pasta Dry ferrous sulfate

Rice Ferric pyrophosphate (2x amount)

Dry milk Ferrous sulfate plus ascorbic acid

Fluid milk Ferric ammonium citrate Ferrous bisglycinate Micronized dispersible ferric pyrophosphate

Cocoa products Ferrous fumarate plus ascorbic acid Ferric pyrophosphate (2x amount) plus ascorbic acid

Salt Encapsulated ferrous sulfate Ferric pyrophosphate (2x amount)

Sugar NaFeEDTA

Soy sauce, fish sauce NaFeEDTA Ferrous sulfate plus citric acid

Juice, soft drink Ferrous bisglycinate, ferrous lactate Micronized dispersible ferric pyrophosphate

Bouillon cubes Micronized dispersible ferric pyrophosphate

Cereal based complementary foods Ferrous sulfate Encapsulated ferrous sulfate Ferrous fumarate Electrolytic iron (2x amount) All with ascorbic acid (2:1 molar ratio of ascorbic acid: iron)

Breakfast cereals Electrolytic iron (2x amount)

Ferrous bisglycinate

Ferrous bisglycinate (used in as a fortificant in the experimental product) is a chelated form of

an iron fortificant. The chelation occurs when amino acids are attached to a mineral. In the case

of ferrous bisglycinate two molecules of amino acid are bound with a covalent bond to an iron

27

molecule. Absorption of this type of iron in the small intestine is similar to that of amino acids:

no irritation or constipation or any other side effects are experienced as with other forms of

iron supplementations. In addition, the mechanism by which this type of iron is absorbed seems

to be determined by blood hemoglobin levels. This is important in preventing toxic levels of

iron in the body.19 Furthermore, it has been reported that losses of vitamins in multivitamin

mixtures caused by amino acid chelates are lower than those caused by ferrous sulphate.139

Ferrous bisglycinate is usually recommended for liquid milk and other beverages: it is classified

under the rubric Generally Recognized as Safe (GRAS).140

The relative bioavailability of iron compounds is articulated by comparing their bioavailability

with ferrous sulphate (relative bioavailability of ferrous sulphate = 100%). Compared to ferrous

sulphate, iron from ferrous bisglycinate chelate (Ferrochel®) has been found to have a 3.4 – 4

times higher relative absorption rate in infants with iron-deficiency anemia,141 iron-sufficient

men142 and anemic adolescents.143 A study by Layrisse et al. (2000),144showed that even in the

presence of iron absorption inhibitors (phytates and polyphenols), the relative bioavailability of

iron from Ferrochel® in non-anemic adults is twice as high as that achieved with ferrous

sulphate. Despite contradictory reports concerning ferrous bisglycinate efficacy145,146 in the

prevention and control of iron deficiency and iron deficiency anemia it has been proved

conclusively in several supplementation trials with infants, preschool children and adolescents

that ferrous bisglycinate can improve the iron status of children.15,143,144

Ferrous bisglycinate appears to be a good fortificant because of its high bioavailabilty and

relatively low reactivity, particularly in milk products.139 Its efficacy in fortified liquid milk,

sweetened bread rolls and whey-based beverage was reported satisfactory in three studies

carried out in Brazil,147,148,149 and also in an iron fortified milk drink trial in Saudi Arabia.150A

South African study on the efficacy of bread made from high-extraction flour fortified with

ferrous bisglycinate, reported a small but significant increase in both hemoglobin and ferritin in

school children.151

28

2.3.2 Education combined with dietary diversification

Dietary diversification which involves nutrition education is a long term strategy for controlling

any micronutrient deficiency. Nutrition education helps to create awareness which has to be

converted into action.105 International strategies customized to South African context such as

Food based dietary guidelines (FBDGs) are part of the nutrition education strategy.24 Dietary

diversification aiming at improving iron status should focus on increasing bioavailability of iron

in the diet through high intakes of enhancers and reduced intake of inhibitors.105However, in

light of challenges that might confront prospective behavior change the purpose in view may be

equally served by employing other strategies such as fortification and supplementation besides

food diversification.105

2.3.3 Supplementation

According to the ADA Report (2005), a supplement is a product (excluding tobacco) intended to

complement the diet that contains a few, or most, or a combination, of the following the

dietary ingredients: a vitamin or mineral; a herb or other botanical; an amino acid; a dietary

substance for human use to supplement the diet by increasing the total dietary intake; or a

concentrate metabolite, constituent or extract.152 It can also be described as a product to be

taken orally in a tablet, gel cap, or liquid form; and as a product that is not meant for use as a

conventional food or a sole item of a meal forming part of a dietary regimen.152

Supplementation should therefore proceed with due consideration of the fact that a healthy

diet should comprise a balanced diversity of foods.152 By the same token, however, it should be

noted that the view commonly held within the ambit of nutritional science to the effect that a

balanced diet can meet all nutritional requirements has been challenged.

For example the Nutrition United Nations Sub Committee on Nutrition has declared that dietary

sources alone cannot provide 100% RDA of micronutrients.153 It is justified to aver, therefore,

that nutritional supplements can play a crucial role in improving physical growth, mental

development, and the prevention of common infections.26

Supplementation can be cost-effective when given to targeted high-risk groups.100Quality

29

control during manufacture and correct dosing are important. Overages are usually included in

the formulation of vitamin supplements during manufacture, to ensure that a certain dosage is

still available by the end of the shelf life. A high- dose Vitamin A supplementation programme in

South Africa is being followed since 2001.24Supplementation seeks to control existing

imbalances which may have pathogenic consequences, such as severe iron deficiency. The

purpose of iron therapy is to increase hemoglobin values. Restoration of iron stores may take

about 4 months because of the lifespan of the red blood cells, which is approximately 120

days.14

2.3.3.1 Iron supplementation

Women of reproductive age and young children have been the main focus for IDA reduction

programmes. However, the recent increase in studies reporting on IDA in school- children has

resulted in a programmatic response for the relevant age group.20 A summary of studies on the

effect of iron supplementation on the iron status of school children is given in Table 2.6. Iron

supplementation has been found to have a positive effect on the Hb concentration in treatment

groups, with more significant changes observed in subjects who were anemic at baseline. This

indicates that iron-replete groups were unable to absorb much iron. Deworming of subjects at

the start of the interventions boosted Hb concentration even in the placebo group, which

explains the importance of eradicating parasitic worms in order to improve iron status.154

30

Table2.6: Overview of iron supplementation trials on iron status of school age children.

Reference Country Initial sample size

Type of study Age group Baseline Hb concentration (g/dL)

Duration/supplement Hb outcome (g/dL)

Seomantri (1989)

155

Indonesia 130 ANPL:24 NAPL:35 ANFe:34 NAFe:37

Double blind randomised clinical trial

8.1 – 11.6 yrs ANPL:9.6 NAPL:13.2 ANFe:9.7 NAFe:13.3

3 mo Iron sulphate 10 mg.kg

.1.d

.1

After treat. 3mo.later ANPL:9.5 ANPL 9.6 NAPL:13.3 NAPL 13.4 ANFe13.0 Ante 13.0 NAFe:13.6 NAFe 13.8 Treatment effect observed in anemic but not non anemic children. Not indication whether outcome was significant or not.

Sungthon et al.(2004)

156

Thailand (397) Daily;140 Weekly; 134 Placebo;123

Double blind, randomised placebo controlled trial

Grade 1 to 6 Daily;12.1 Weekly; 12.2 Placebo;12.1

16 wks Ferrous sulphate 30.0mg

Daily;12.8 Weekly; 12.7 Placebo;12.5 A positive treatment effect was observed

Rochnick et al.(2004)

157

Phillipines 1510 Interv: 708 Con: 802

Randomised controlled trial

7 – 12 yrs Intervention:12.4 Control:12.6

17 wk Ferrous sulphate 32.5mg

Intervention:12.4 Control:12.2 Hb concentration of children in intervention group did not change significantly. Hb for untreated group fell.

Hb: hemoglobin; ;ANPL:anemic placebo treated group ; NAPL:non anemic placebo treated group; ANFe:anemic iron supplementation treated group ; NAFe:non anemic iron supplementation treated group.

31

Best absorption of iron supplements is achieved on an empty stomach. However,

gastrointestinal side effects such as nausea, epigastric discomfort and distention, heartburn,

diarrhea or constipation may reduce tolerance and compliance. Oral iron supplements such as

ferrous iron salts (ferrous sulphate and gluconate) are usually preferred because of their low

cost and high bioavailability. Other supplements include: amino acid chelated ferrous

bisglycinate, synthetic chelated NAFerredetate, and EDTA (ethylenediaminetetraacetic acid).

However, the efficacy of ferrous bisglycinate has proved superior to that of its rivals and less

prone to produce undesirable side effects.19

The studies show that iron supplementation on its own has a positive effect on the iron status

of school age children. Significant changes were particularly noticeable in anemic children.

2.3.3.2 Multiple micronutrient supplementation or fortification

Micronutrient deficiencies have been observed to overlap and occur simultaneously in the

same group of people.20 Provision of multiple micronutrient supplementation or fortified foods

to the affected or vulnerable groups may therefore be cost-effective in addressing nutrient

deficiencies.20

A summary of studies on the effect of micronutrient supplementation on the iron status of

school children is provided in Tables 2.7 and 2.8. The latter gives detailed information on the

nutritional content of the supplements used in the studies summarised in Table 2.7. The

intervention/treatment groups involved in these randomised controlled trials were given foods,

beverages, seasoning, biscuits, bread or tablets enriched with multiple micronutrients, whereas

the controls received placebo and/or iron supplements alone. The treatment groups showed an

increase in Hb concentration at varying quantities. The conclusion favouring iron

supplementation alone in some instances, especially in virtue of apparent lack of increase in Hb

after the multiple micronutrient intervention, was most probably drawn as a result of

measurement error.160 It is also important to note the possible effect due to the variability in

the duration of the intervention studies, some had a duration as short as 8 wks158 while some

went on for as long as 8 to 12months161,163,65,166.

32

A systematic review of randomized studies on the placebo effect on Hb response, compared to

that of combining multiple micronutrient with iron supplementation, showed a significant

increase in Hb concentration in children’s weight mean difference (WMD) = 0.65 g/dL, 95% CI

0.50, 0.80, P < 0.001). An initial greater rise was seen in anemic children and in children in the

lower ranges of height-for-age z-scores.159

A pooled analysis of studies comparing combined Fe and micronutrient supplementation with

Fe supplementation alone showed that the addition of multiple micronutrients to Fe resulted

in a small but significant increase in Hb (WMD = 0.14 g/dL, 95% CI 0.00, 0.28, P = 0.04) over Fe

supplementation alone.159 According to expectation, therefore, synthesized evidence alone

shows that instead of impairing the Hb response to iron supplementation children, a judicious

addition of multiple micronutrients may have marginal benefits compared to iron

supplementation alone.159 However, given the mixed results of previous studies, interaction

may be more likely with high dosages of micronutrient supplementation and shifting from a

single to multiple micronutrient supplementation may therefore still have to overcome

challenges such as deficient programme efficacy.159

A food based strategy involving enriched food products, for example, nevertheless remains a

promising nutritional intervention. However, more evidence on the efficacy and effectiveness

of this type of intervention is needed for policy and programme planners to have it

implemented.62

33

Table 2.7: Overview of multiple micronutrient supplementation/ fortification trials on mean Hb concentration in school age children

Hb : Haemoglobin , Praziquantel (P) , Praziquantel + Iron (P+Fe) , Praziquantel+multiple micronutrient supplementation(P+MM) , Praziquantel +iron +multiple micronutrient supplementation (P+Fe+MM), Iodized salt(IS), Triple fortified salt(TFS).

Reference

Country Sample size Type of study Age group Baseline Hb concentration (g/dL)

Duration Intervention Hb outcome (g/dL)and comments

Ayoya et al (2009). 162

Mali

847

Randomised controlled trial

7 – 12 yrs

10.37 (P) 11.42 (P+Fe) 10.57 (P+MM) 10.59 (P+Fe+MM)

12wk

Praziquantel (P) Praziquantel + Iron (P+Fe) Praziquantel+multiple micronutrient supplementation(P+MM) Praziquantel +iron +multiple micronutrient supplementation (P+Fe+MM)

11.54(P+Fe) 10.81 (P) 11.28(P+MM) 11.35 (P+Fe+MM) Possible explanation for low effects of MM on Hb, could be negative interactions among nutrients that interfered with the use of iron or other erythropoietin nutrients

Zimmerman et al. (2004) 58

Morocco

157 goitrous school children with vitamin A and iron deficiency

Randomsed double blind trial

10-13yrs

Iodized salt(IS): 11.6 Triple fortified salt(TFS): 11.4

10 mo

Triple fortified salt

I S: 11.5 TFS: 12.9 Triple fortification of salt effective in increasing hemoglobin levels, possibly because most of the children were taking three main meals plus to snacks per day, all of which had some salt. Thus iron absorption was enhanced by repeated delivery of small doses throughout the day.

vanStuivernberg et al. (2006)151

South Africa

160

Randomised controlled trial

6-11yrs school children

Control:12.7 Electrolytic iron:12.6 Ferrous bisglycinate:12.7

7.5 months

Fortified bread

Control:12.8 Electrolytic iron:12.7 Ferrous bisglycinate:12.9

34

Table 2.7:(cont.) Overview of multiple micronutrient supplementation/ fortification trials on mean Hb concentration in school age children

Reference Country Sample size Type of study Age group Baseline Hb concentration

(g/dL) Duration Intervention Hb outcome

(g/dL)and comments

Osei et al. (2010)163

India

499

Randomised control trial

1-8 yrs

Micronutrient premix fortified: 12.2 Non fortified: 12.17

8 mo

Micronutrient premix added in lunch meals Nonfortified lunch meals

Micronutrient premix fortified: 12.32 Nonfortified: 12.25 Slight increase in Hb of boh groups

Jinabhai et al. (2001) 164

South Africa

579

Double blind randomised placebo controlled trial

8 – 10 yrs

Vit A +iron grp;12.8. Vit A group;12.7. Non fortified group;12.8

16 wks

Fortified biscuit

Vit A +iron grp;12.9. Vit A group;12.8. Non fortified group; 12.9. No treatment effect, may have been due to low prevalence of anemia at baseline.

vanStuijvernberg et al. (1999) 167

South Africa

Experimental; 115 Control;113

Randomized controlled trial

6 -11 yrs

Intervention grp; 12.5 Control grp;12.6

3 wks over a 12 mo period

Fortified biscuit and cold drink

Intervention grp; 6 mo; 12.4 12 mo; 12.9 Control grp; 6 mo; 12.4 and 12 mo; 12.7

Hb: hemoglobin, Vit A: Vitamin A, mo: month, grp: group

35

Table 2.7 (cont.) Overview of multiple micronutrient supplementation/ fortification trials on mean Hb concentration in school age children

Reference

Country Sample size Type of study Age group Baseline Hb Conc (g/dL)

Duration Intervention Hb outcome (g/dL)and comments

Abrams (2003) 158

Botswana

311 Exp:164 Con:147

Non random clinical trial

6 -11yrs

Exp: 12.9 Con: 12.9

8wk

Fruit flavoured fortified beverage

Exp: 12.6 Con: 12.2 Changes in Hb significantly different between experimental and control group. Reduction in Hb level may have been due to change in measuring equipment

Taljaarrd et al.(2013)168

South Africa

CNS;103 CS;104 MNNS;103 MNS;104

Randomised double - blind, controlled intervention

6 -11yrs

CNS; 12.7 CS;12.7 MNNS;12.5 MNS;12.7

8.5 mo

A beverage with and without micrnutrients

CNS; 12.7 CS;12.7 MNNS;12.9 MNS;13.0

Ash et al. (2003)132

Tanzania

841

Randomized double blind placebo controlled

6 -11 yrs

11.9 (MM fort. bev) 11.9 (non fort bev)

6 mo

Multiple micronutrient fortified beverage

11.6 (MM fort. bev) 11.2 (non fort bev) Hb decrease in both groups due to seasonal influence on dietary quality and morbidity pattern with regards to malaria.

Hb: hemoglobin, Ex: experimental group; Con : control proup; CNS : no micronutrients (control beverage) with non nutritive sweetener , CS : no micronutrients(control beverage) with sugar, MNNS : micronutrients with a non nutritive sweetener, MNS: micronutrients with sugar, mo: months, MM fort bev: multiple micronutrient fortified beverage, non fort bev: non fortified beverage.

36

Table2.8: Nutritional content of the supplements /fortified foods used in the multiple micronutrient studies Nutrients 158 132 162 58 163 164 167 168 151

Vitamin: A (ug) (RE)

B carotene 2400

Retinyl palmitate 1750 IU

Vitamin acetate 1030

60 30 350 B carotene 2.0

257.2

Thiamin(mg

1.5 0.25 0.28

Riboflavin (mg)

0.4 0.6 1.7 0.26

Niacin (mg)

2.7 20 3.41

Pyridoxine (mg)

0.5 0.7 400 0.38

Folic acid (ug) 14 0.14 10 1: 0.1 2: 0.2

206

B12 (ug)

3

Biotin (ug)

30

Ascorbic acid (mg)

60 72 120 110

Pantothenat 10 Cynocobalamin (ug)

1.0

Tocopherol (mg)

7.5 10.5 23

Calcium (mg)

250

Iron (mg)

Ferrous bisglycinate chelate 7.0

Ferrochel 5.4 Ferrous fumerate 18

Fe PP 2 Ferrous sulphate 3mg of elemental iron.kg body weight.1.d.1

FeEDTA 5 Fe fumerate 5.9 1.EleFe 20 2.eleFe 40 3. Fe sul30 4.eleFe 60

Elect: 5.04 Ferro bisgly:5.04 Contr;±1.8

Magnesium (mg)

100

37

Nutrients 158 132 162 58 163 164 167 168 151

Iodine (ug)

60 45 150 95.4

Phosphorus (mg)

77

Zinc (mg)

3.75 5.25 15 2.5 2.16

Selenium (ug)

25

Potassium (mg)

40

Molybdenum (ug)

25

Boron (mg)

150

Chloride (mg)

36

Nickel (ug)

5

Copper (mg)

2

Chromium (mg)

120

38

2.4. LITERATURE REVIEW SUMMARY

Anemia is most prevalent in developing countries (i.e. 39% of children aged < 5years, 48% of

children aged 5-14 years, 42% of all women and 52% of expectant women). Iron deficiency

accounts for about 50% of anemia.97Iron deficiency anemia is the final stage of iron deficiency

and is characterised by low hemoglobin levels.105 Factors that may contribute to the

development of iron deficiency include low intake and poor absorption of iron from the diet

due to the presence of iron absorption inhibitors or lack of absorption enhancers; and a greatly

heightened physiological need for iron during menstruation, pregnancy and growth.37,38 It is

common cause that measurement of hemoglobin, ferritin and sTfR in conjunction with chronic

infections as further indices, produces the best results when assessing iron status. However,

this procedure is usually expensive and difficult.94 Hemoglobin measurement has therefore

been successfully substituted as an alternative method in situations challenged by financial

constraints and in remote field works.96

There is good evidence that Iron deficiency anemia can cause retarded physical development,

low cognitive function, weak immune function, growth decline, and accelerated fatigue.106

Micronutrient deficiencies have been observed to overlap and occur simultaneously in the

same group of people.20 Provision of multiple micronutrient supplementation or fortified foods

to the affected or vulnerable groups may therefore be cost effective in addressing nutrient

deficiencies.20

39

CHAPTER 3: METHODOLOGY

3.1 ETHICAL CONSIDERATIONS

Ethical approval to undertake this study was granted by the Research Ethics Committee of the

Faculty of Health Sciences (University of Pretoria) (Addendum 1). Permission to conduct the

study was given by the Gauteng Department of Education and the Sunnyside Primary School.

Informed consent was also sought and granted by the parents or legal guardians of the

learners. All aspects of the protocol were explained to the learners in their classrooms. Learners

with moderate to severe anemia (Hb < 9.5) were not included in the study were referred for

treatment.

3.2 RESEARCH DESIGN

An experimental study design in the quantitative domain was used. The study was a

randomised double blind placebo controlled trial (Figure3).

3.3 STUDY SETTING

Sunnyside Primary School in Sunnyside, Pretoria, situated within the municipal confines of

Tshwane Metro Council (Gauteng Province, South Africa).

3.4 STUDY POPULATION

The study population consisted of male and female learners aged 8 – 12 years enrolled for

Grades 3 and 4 the academic year 2010.

3.4.1. Recruitment and screening

Parents or guardians of learners enrolled for Grade 3 and 4 at Sunnyside Primary School were

informed during a scheduled general parents meeting about the study and its purpose, and

were given an opportunity to ask questions. Children whose parents/guardians had signed

consent forms for participation in the study were eligible for participation in the study and had

to sign assent forms (Addendum 2). Screening was done and children who met the inclusion

criteria were enrolled in the study.

40

Figure 3. Trial profile of the 14 weeks intervention study.

Inclusion criteria

_ Boys and girls in Grades 3 and 4

_ English literate

_ Children with normal iron status (Hb > 11 g/dL) and mild anemia (Hb > 9.5 g/dL)

Group B - Experimental n= 73

Group A - Control n= 71

Completed the study n= 73

Completed study n= 64

Dropouts n =7 Reason: Instructed by parents

All eligible learners Grade 3&4 (8-12yrs)

Deworming

Randomised according to grade, class gender and Hb

Dropout n = 0

Iron status n =144

41

Exclusion criteria:

_Children with moderate to severe anemia (Hb<9.5 g/dL )

3.5 SAMPLING METHOD

A convenient purposive sampling method was used for this research study. A primary school

with a need for school feeding and with an existing school feeding program in place was

chosen. Convenient sampling was used for logistical ease.

3.5.1 Sample size

Sample size was calculated based on the comparison of the two groups with respect to their

change in Hb following 14 weeks of intervention treatment. This was done with either

micronutrient enriched or none micronutrient enriched maize based liquid meal supplement. A

difference in change from baseline of 0.8 g/dL between the groups was regarded as clinically

significant and a standard deviation of 1.5g/dL was assumed (range/4 = (15.5 – 9.5)/4 =1.5g/dL).

For a one sided test at the 0.05 level of significance a sample of 61 subjects per group had a

90% power to detect the clinically significant difference of 0.8g/dL. To account for a dropout

rate of 30%, a sample size of 80 subjects per group was aimed for, however, only 71 and 73

learners were included, allowing for a dropout rate of about 20%.158

3.6. INTERVENTION

3.6.1 De-worming

All participating children were dewormed before the start of the intervention to eliminate

parasitic infestation which results in chronic intestinal blood loss due to the release of

anticlotting agents.56 A single dose of mebendazole (Vermox 500 mg tablet) was administered.

3.6.2 Experimental product

3.6.2.1 Description

The meal supplement used in the study was produced and provided by a South African based

company supplying locally manufactured maize-based ready-to-use “TetraPak” packed meal

supplements. Unfortified maize as the staple food of the target population was used as the

foundation of the products.The meal supplement was a lactose free, gluten free enteral feed,

42

providing 1 kcal/ml and 9 g protein per 250 ml Tetra-Pak portion. The meal supplement was

fortified with micronutrients and had the following nutritional specifications:

High in energy (>250 kJ per 100 ml)

High in vitamins (A, D, E, C, B1, B2, Niacin, B6, B12, Biotin & Pantothenic acid)

High in minerals: calcium, phosphorus, iron, magnesium, zinc & iodine

Source of protein (>2.5 g per 100 ml and >2.5 g per 418 kJ)

High in carbohydrates (>6.5 g per 100 ml)

The control meal supplement had a similar macronutrient profile but without any added micronutrients (Table 3.1).

43

Table 3.1: Nutritional composition of experimental and control product for boys and girls

Nutrient Experimental product

%RDA(9yrs) %RDA(12yrs) Control product % RDA

Energy 1050 KJ 11 1050 KJ 11

Protein (g) 9 26 16 9 26

Carbohydrate (g)

32.5 32.5

Fat (g) 10 10

Fibre-inulin FOS(g)

3 3

Sodium (mg) 275 275

Potassium (mg) 375 375

Chloride (mg) 355 355

Vit A (ug RE) 452.5

65 45

Vit D (ug) 2.5 25 50

Vit E (mg) 9.5 136 95

Vit C (mg) 45 100 75

Vit B1(mg) 0.8 67 57

Vit B2 (mg) 1 71 63

Niacin (mg) 15 94 83

Vit B6 (mg) 1.5 94 75

Folic Acid (ug) 50 17 25

Vit B12 (ug) 0.75 25 75

Biotin (ug) 50 42 50

Pantothenic acid (mg)

4.88 98 81

Calcium (mg) 325 41 41

Phosphurus (mg)

312.5 39 39

Iron (mg) 5 63 63

Magnesium (mg)

105 42 35

Zinc (mg) 5 50 33

Iodine (ug) 100 83 67

Selenium (mg) 0.13 65 -

44

Other characteristics of the product include: Gluten and lactose free; prebiotics (inulin), all

essential amino acids and non-essential amino acids. The product incorporated bioavailable

amino acid chelated minerals including ferrous bisglycinate chelate (Ferrochel®) whose

bioavailability was found to be far superior to the iron from ferrous sulphate.

The maize-based liquid meal supplement was a product used for the following conditions:

Full fluid liquid diet

Remedial treatment for malnutrition, underweight and micronutrient deficiencies

Immuno-compromised conditions, e.g. HIV & AIDS

Tuberculosis

Gastro-intestinal disease, e.g. diarrhea, Irritable bowel syndrome

Supplementation in addition to meals

Poor appetite

Cerebral palsy

Mental health conditions

All other clients as identified by a health professional

The product came in three different flavours (vanilla, chocolate, banana). The vanilla-flavoured

product was used in the study under review.

3.7 RANDOMISATION AND BLINDING

Learners in each class were stratified according to gender and baseline haemoglobin levels then

randomly assigned to two groups (Group A and Group B). A double blind study design was used

for this study. To prevent bias neither the subjects nor the investigator assessing the response

were told of the treatment the subjects were receiving.

3.7.1 Preparing and administering of experimental product

As a "ready to drink" product the maize-based liquid meal supplement did not require any

further preparation before uses. The pre-portioned meal supplements were numbered before

being given to the learners. Supplements intended for Groups A and B were marked with

numbers to be administered accordingly to specific subjects. Children were given supplements

each morning before 10 am. Each learner produced an identity tag before she/he could be

45

given the drink allocated to him/her by number as indicated. The said numbering by subject

was employed as a safeguard against dispensing product twice to a specific subject.

3.7.2 Subject compliance and monitoring

Each child was given an identity tag with the subject number, name, and class (reflecting the

relevant school grade) on it. These identity tags were yellow for Group A and blue for Group B

(Addendum 3). The researcher and assistant monitored compliance by observing the children as

they imbibed their drinks, making sure they did not share. Empties or leftovers were collected

and records of attendance and amounts left over were weighed and recorded in grams on

compliance sheets (Addendum 4) and filed. Compliance or consumption was expressed as

portion percentage (%).

3.7.3 Packaging and blinding of experimental products

The packaging and labeling of the two products were identical. The expiry date printed in lower

cases on the upper surface of the packaging had different dates for each product, and was the

only mean of differentiating between the two products.

3.8 DATA COLLECTION

3.8.1 Screening

Screening of the learners for eligibility to participate in the study was done in July 2010.

Standardised procedure was employed to measure Hb with the aid of a portable HemoCue

photometer (HemoCue Hb 201+Analyser, Angelhom, Sweden).123 Adjustment to hemoglobin

cutoffs was carried out according to altitude and ethnicity (Table 2.5).18 The adjustment led to a

Hb cutoff of 11.0 g/dL for the school children serving as subjects. Learners whose Hb was above

9.5 g/dL were included/invited to participate in the study.

3.8.2 Schedule of measurements

Weight, height, and Hb measurements were taken at the beginning of the intervention (July

2010), and again at the end of the intervention (November 2010).

46

3.8.3 Variables

3.8.3.1 Iron status

The assessment of iron status was achieved by using hemoglobin as the biomarker. No other

parameters were used due to financial and ethical constraints. Baseline and end assessments

were carried out by qualified dietitians and trained dietetic students. Hemoglobin

concentrations were determined with the aid of a portable HemoCue photometer (HemoCue

Hb 201+Analyser, Angelhom, Sweden). The accuracy and precision of hemoglobin values

measured with the aid of the HemoCue photometer are comparable to those obtained with the

cyanmethemoglobin method (the most reliable method recommended by the International

Committee for Standard Hematology). Finger pricks were done by using a single-use lancing

device (Accu-Check® Safe-T-Pro Uno). This lancing device is safety engineered with safety wings

that break during the use to prevent re-use, avoid accidental finger pricks, and eliminate cross-

contamination.103

3.8.3.2 Anthropometric measurements

Anthropometric measurements were recorded at two different time-points (baseline and end)

according to standard techniques by trained dietitians and dietetics students.96 Measures

included height and weight. Body mass index (BMI) for age and height for age were expressed

as z- scores.169

Height

Height was measured with a portable stadiometer (the Leicester height measure, England max

height 2.10 m). In measuring height, clothes were minimal so that posture could be clearly

seen and shoes were taken off. Height measurement was taken using standard techniques to

the nearest 0.1 cm.96

Weight

Weight was measured with the aid of a digital personal scale (Body – Check Analysis- Seca sense

804, Germany). The scale was placed on a hard flat surface (not carpet), checked and adjusted

for zero balance before each measurement. Body weight was measured according to standard

techniques and recorded to the nearest 0.1 kg.96

47

Z -scores

Z-scores for height-for-age (HAZ) and BMI-for-age (BAZ) were determined with the aid of the

WHO Anthro Plus Software.169A HAZ (z-score < -2SD) was indicative of moderate stunting and

HAZ < -1SD was indicative of mild stunting. On the other hand a BAZ < -3 SD reflects severe

thinness, BAZ <-2SD indicated thinness, and BAZ > +1SD indicated overweight and BAZ >+2 SD

reflected obesity.

3.8.3.3 Socio-demographic information

Socio-demographic information was solicited from participants in a standard, made-to-measure

questionnaire (Addendum 5). Parents/guardians were asked to fill in the questionnaires and

return them through their children.

3.8.3.4 Sickness diary

Children were given a sickness diary, to be filled in by their parents whenever they suffered a

bout of illness during the period of study (Addendum 6). The diaries were collected at the end

of the study.

3.9 STATISTICAL ANALYSIS

This randomised, controlled trial was conducted to compare the Hb levels of children aged, 8 –

12 years in experimental and control groups. Only subjects with both baseline and end data

were included in the statistical analysis.

The statistical software STATA Release 11 was used for statistical analyses.

Descriptive statistics were used for all measurements. Groups were compared with respect to

change in Hb based on an analysis of covariance (ANCOVA) with baseline Hb values as

covariate. Testing was done at the 0.05 level of significance.

48

CHAPTER 4: RESULTS AND DISCUSSION

4.1 CHARACTERISTICS

4.1.1 Socio-demographic data

Of the 144 learners who started as participants in the study 137 (95%) remained in it for the

duration of 14 weeks. Seventy three (73) were in Group B (experimental group) and 64 in Group

A (control group) (Table4.1).

Table 4.1: Characteristics of learners who participated in the 14 week study

Experimental group

(n= 73)

Control group

(n=64)

Male: n (%) 31 (42)

28 (44)

Female: n (%) 42 (58) 36 (56)

Mean age (years) 9 ( 0.8) a 9 ( 0.9) a

a standard deviation

Data affecting socio-demographic characteristics (Table 4.2) were obtained from 49% of the

participants who remained in the study for the duration, while 51% did not return the

questionnaires. As noted by Keskin et al.(2005), a high prevalence of iron deficiency anemia

correlates closely with low socio-economic status (SES).134 It was noted that, the consumption

of tea was high in the low socio-economic groups, whereas the consumption of red meat, fish

and citrus fruits tended to be lower by comparison in that group.134

Children whose parent/guardian (head of household) had no education, a low income and no

car, were considered to be of low socio-economic status in Keskin et al.’s study.134 However, the

findings (Table4.2) in this study were to the positive, meaning the children were of high SES.

Therefore, it can be assumed that the low prevalence of anemia among the children in this

study was attributed to their high SES.

49

Table 4.2: The socio-demographic characteristics of the study population (n = 67)

Characteristic N %

Size of family

2 6 9 3 9 3

4 19 28

5 16 24 6 6 9

7 7 10

8 4 6

Mother’s marital status

Unmarried 14 21

Married 41 62 Divorced 3 5

Separated 3 5

Widowed 3 5

Traditional marriage 2 3

Who cooks food?

Father 2 3

Mother 54 81 Sibling 5 7

Grandmother 2 3

Aunt 3 4

Other 1 1

Who buys food?

Father 1 1

Mother 59 88 Sibling 3 4

Grandmother 2 3

Aunt 2 3

Head of household Father 34 51

Mother 27 40

Grandmother 2 3 Aunt 2 3

Friend 1 1

Other 1 1

Who decides how much money is spent on food?

Father 13 20

Mother 50 76 Grandmother 2 3

Aunt 1 2

Mom’s level of education

None 2 3 Primary school 4 6

Std 6-8 1 2

Std 9-10 18 3 Tertiary education 37 57

Not applicable 3 5

Household income

None 3 4 R100-500 2 3

R500-1000 5 7

R1000-3000 12 18 Over 5000 15 22

50

4.1.2. Anthropometric characteristics

Anthropometry determines either the cross-sectional nutritional status of a population, or the

nutritional status of an individual.109 The anthropometric characteristics of participants in the

study under review are presented in Table 4.3. There was no significant difference in the weight

of the experimental (33.6 ± 9.6kg) and control (35.3 ± 9.0kg) (P= 0.281) groups at baseline. Both

groups gained some weight, though not significantly, during the period of the study. There was

also no significant difference in the height of the groups at baseline. The participants were not

stunted. The mean BMI z-scores indicated that the learners were not obese. These findings

indicate that there was no significant difference in the nutritional status of the participants at

baseline.

Genetics and the environment play a major role in influencing physical growth. The

environment, and the quality and quantity of food available are important determinants of

growth rate.170,171 The NFCS showed that one in ten of all children aged 1 – 9 years were

underweight, more than one in five were stunted and 6% of children were overweight. The

same survey found that the prevalence of stunting declined while that of excess body weight

rose as maternal education levels increased.109 Although the average age of children involved in

the study was slightly higher than that of their counterparts in the national survey, their

average age (9.8 years) nevertheless fell within the same age bracket as the said counterparts,

therefore comparison of the survey results with those of the present study is justifiable. Thus,

as in the national survey, few participants (< 10%) were underweight.109,172 By contrast,

however, the incidence of stunting and overweight recorded for the survey was different from

that recorded in the study at issue.

In particular, stunting was lower among participants in the present study (< 10%) compared to

the moderate prevalence recorded in the NFCS (20 – 29.9%). Contrariwise, the prevalence of

overweight participants was higher (> 20%) in the present study than that reported by the NFCS

(6%).109 The results emanating from this study were comparable to those of the South African

National Health and Nutrition Examination Survey (SANHENS), which showed that the

prevalence of undernutrition had declined among children of all age groups in South Africa.128

51

Table 4.3: Anthropometric characteristics of the study population

HAZ: Height for age z-scores; BAZ: Body mass index for age z- scores Stunted (HAZ < -2); Wasted (BAZ < -2); 1(ANCOVA, P = 0.05)

At the end of the study no significant changes had become evident in the anthropometry of the

participants (Table 4.3). Other studies undertaken with school children showed significant

increases in growth as well as height-for-age and weight-for-age z- scores.112,113 The positive

results recorded in these studies were attributed to participant’s low iron stores at baseline.

Results that seemed to vary erratically in the instance of studies conducted with iron-replete

children could have been attributable to variations in administered iron dosages, coinciding

Variable Experimental group (n= 73)

Control group (n=64)

p- value1

Mean (sd) Mean (sd)

Weight (kg)

Baseline 33.6 (9.6) 35.3 (9.0) 0.281

End 34.6 (10.1) 36.5 (10.1) 0.276

Height (cm)

Baseline 136.2 (7.8) 137.9 (9.3) 0.255

End 137.7 (0.9) 139.6 (1.3) 0.219

HAZ (stunting)

Baseline 0.009 (1.08) 0.265 (1.20)

0.153

End 0.037 (1.095) 0.292 (1.249)

0.152

Change from baseline to end

0.028 (1.082) 0.027 (1.198) 0.237

BAZ (wasting)

Baseline 0.442 (1.18) 0.650 (1.18)

0.309

End 0.321(1.352) 0.537(1.302)

0.367

Change from baseline to end

0.121 (1.181) 0.113 (1.177) 0.704

52

deficiencies, studies running over varying length of time or combinations of these factors.116

4.1.3 Consumption

The maize-based liquid meal supplement enriched with multiple micronutrients as used in the

study under review was provided for a total of 69 school days over a period of 14 weeks

(shortened due to strike action). Mean consumption (defined as the actual amount of drink

consumed during the study, expressed as a percentage of the total amount provided over the

trial period) was 49.34 % (SD: 27.43) and 53.71% (SD: 31.94) in the experimental and control

groups respectively. The difference in consumption of the drink between the two groups was

insignificant (P = 0.394)

The experimental product provided 5 mg iron per day providing 63% DRI of iron intake when

consumption is complete. However, since the consumption was 49.34% the average daily

intake per individual can be assumed to have been 2.47 mg, thus amounting to 30.9% of DRI

per day. The school children took the maize-based liquid meal supplement in the early hours of

the day, which probably meant that they had taken the supplement shortly after consuming a

breakfast meal, which would therefore not have been fully digested by the time they ingested

the supplement, thus possibly accounting for the low consumption recorded in the course of

the study. Other possible reasons for the low consumption levels may have been absenteeism,

unavailability of child during allocated scheduled handout of supplements, and the observed

relative distaste for the experimental products. Consumption overall as observed in the study

under review is lower than that found in comparable studies.

A consumer sensory evaluation (data not included) of the same product showed that the

learners preferred (not markedly though) the chocolate flavoured maize-based liquid meal

supplement enriched with multiple micronutrients to the other two flavours (vanilla and

banana).173 This preference could be the reason why consumption as observed in this study was

low.

53

4.1.4 Iron status

The prevalence of anemia at baseline in the experimental and control groups is shown in Table

4.4. In the experimental group six (6) cases of mild anemia were diagnosed at baseline (Hb <

11g/dL) while only one (1) participant in the control group was diagnosed as mildly anemic at

that stage. By the end of the study, however, only one (1) participant in the experimental group

had mild anemia, while the control group still had one (1) mildly anemic participant.

This was not unexpected as the learners in the control group were not getting any

micronutrient from their product. Table 4.5 shows the mean Hb levels of the experimental and

control groups. There was no significant difference between the experimental and control

groups in the Hb levels (12.6 ±1.1 g/dL in the experimental group and 12.8±1.1 g/dL in the

control group) (P = 0.250) at baseline (Table 4.5). It should be noted however that Hb was used

in the study as an inexpensive and common measurement, otherwise when used alone Hb is

not a very specific and sensitive indice to determine whether iron deficiency is the specific

cause of anemia.96

Table 4.4: Anemia prevalence in the experimental and control groups at baseline and at end

The intervention had no significant effect on participants’ Hb levels over the intervention

period of 14 weeks. However, at the end of the trial a slight increase in Hb levels (0.08 ±

1.210g/dL) was found in the experimental group, while a decline (-0.249 ± 1.191g/dL) was

observed in the control group (Table 4.5); moreover the prevalence of mild anemia (Hb<

11g/dL)) in the experimental group had decreased from 8% at baseline to 1%.

Mild anemia (Hb 9.5 to ≤

10.99g/dL)

Experimental n(%) Control n(%)

Baseline 6 (8) 1(2)

End 1(1) 1(2)

54

Table 4.5: Iron status of participants at baseline, end and change from baseline to end

Experimental

Mean ± sd

Control

Mean ± sd

p-value1

Baseline Hb (g/dL)

12.6 ± 1.1

12.8 ± 1.1

0.250

End Hb (g/dL)

12.7 ± 0.12

12.6 ± 0.11

0.806

Change from baseline to end

0.087 ± 1.210 -0.249 ± 1.191

0.477

Hb: Hemoglobin; sd: standard deviation; 1(ANCOVA, P = 0.05)

The prevalence of anemia declined in the course of this study from a level regarded as a mild

public health problem (10%)128 to an almost negligible level (2%) despite the low mean

consumption observed (50%). An improvement was mainly observed in the experimental

group. Table 4.6 shows that a negative change irrespective of rate of intake of the product was

observed in the iron status of learners in the control group (-0.241 ± 1.258 in members whose

consumption was < 50% and -0.241 ± 1.191 in members whose consumption was ≥ 50%). On

the other hand a positive change (0.008 ± 1.097) was evident in members of the experimental

group whose consumption was < 50% and 0.123 ± 1.301 in members whose consumption was ≥

50%. On balance, though, the changes were negligible.

Assimilation of iron is low when iron status is at repletion levels.174 If a high bioavailability diet is

followed absorption averages at 15% in non-anemic individuals but climbs to 50% in anemic

subjects .174 The fact that 90% of the children in this study had normal Hb levels (Hb > 11g/dL)

could be the main reason why the experimental product used in this study, despite its high

highly bioavailable iron content, made no appreciable difference to subjects’ iron status. Iron

absorption improves when taken on an empty stomach or between meals. The liquid meal

supplement used in the instance under review was taken early in the day when subjects’

breakfast probably had not had time to digest, with the result that intake of the supplement as

well as absorption of its iron content were low.

Results were comparable with those obtained in other studies conducted with subjects whose

55

iron status was at repletion levels. For instance, no observable result materialized from

administering fortified biscuits during an intervention conducted over a period of 16 weeks in

iron replete (12.8 g/dL) school children aged 8 – 10 years old.164 Supplementation also had no

appreciable effect in the instance of another intervention study carried out in Tanzania132

where participants’ baseline iron status had been normal (11.9 g/dL for both the experimental

and control groups). Seasonal influence in dietary quality and the malaria-related morbidity

pattern were given as reasons for the insignificant results achieved by conducting the

intervention. This shows that iron repletion may not be the only reason for insignificant results

obtained with supplementation venture.

The mean age of participants in the study under review was 9 years, but their age range was 8 –

12 years, which means that some were pre-adolescents or even adolescents who might have

started menstruating, in which case the iron requirements of the menstruating female

participants would have been higher than those of other participants and might not have been

met in full by the supplementation.(not assessed).

Since the experimental product utilized in the study under review contained other chelated

micronutrients besides chelated ferrous bisglycinate (Table 3.1), it follows that some of these

additional nutrients may have interacted biologically with iron supplement because they (the

ferrous and other nutrients) have chemically similar absorption and transport mechanisms (e.g.

calcium).175 A number of interactions between micronutrients could take place when a high

dose of a single nutrient is given or when the supply of an individual micronutrient is

inadequate.175 In such instances iron indicators do not improve as greatly as when iron is given

alone.175 Such interaction could have served to suppress the efficacy of supplementation and

could therefore have been partly responsible for the observed insignificant effect of

supplementation over the study period. Moreover, no indication was found in the observed

outcome of supplementation that vitamin C had enhanced iron intake as might be expected.

Similar results were observed in a study by Ayoya162 where a multiple micronutrient

56

supplement administered to school children aged 7- 12 years old schoolchildren (baseline Hb:

10.42 g/dL; 10.57 g/dL; 10.59 g/dL for the Fe, MM, and Fe+MM groups respectively), barely

lifted Hb values over a study period of 12 weeks, as opposed to when iron supplementation was

taken alone. Negative interactions among nutrients were reported to have interfered with the

use of iron or other erythropoietin nutrients. In another study conducted with Botswana school

children aged 6 – 11 years over a period of 8 weeks (baseline Hb:12.9 g/dL for both the

experimental and control groups), it was found that administering a fortified, fruit-flavoured

drink over the study period had resulted in lowered Hb levels158 possibly because in this

instance too, negative interaction had occurred between nutrients. By contrast, however, other

studies involving multiple micronutrient supplementation have reported a positive effect on the

anemia status of the population concerned.

A positive effect for instance, was observed in a study done in Morocco by Zimmerman58 where

it was found that the Hb levels of school children aged 10 – 13 years had been raised by

administering triple fortified salt (baseline Hb: 11.4g/dL and 11.6 g/dL for the experimental and

control groups respectively). The likely reason for these results may have been the fact that the

children were taking three meals per day plus snacks, all of which contained triple fortified salt.

Iron absorption was therefore enhanced by repeated delivery of small doses throughout the

day,58 unlike Zimmerman’s study, the experimental product in this study was only given once a

day, therefore there was no repeated delivery of the micronutrients.

A significant increase in Hb levels as well as high consumption levels ( >90%) were reported by

Van Stuijvernberg’s study in which South African school children aged 6 -11years old (baseline

Hb: 11.5g/dL and 11.3 g/dL for the experimental and control groups respectively) were given

biscuits to eat that had been fortified with multiple micronutrients.167 The high consumption

meant that, unlike the results obtained with the low consumption of participants in the present

study, the children absorbed useful amounts of the nutrients as result of the continuity.

Positive results on the bioavailability of ferrous bisglycinate have been shown in several studies.

57

For example, in a study conducted in Valencia (Carabobo State, Venezuela) on men aged (15 –

50 yrs old) and women, of whom 40 were menopausal, showed that Ferrochel could partially

prevent the inhibitory effects of phytates, mainly because it is highly soluble even at pH 6,

highly assimilable and not inclined to interact with food.144 The same effect was expected in the

present study as the basis of the liquid meal supplement was maize which contains some

phytates

It is understandable therefore, that the children did not absorb useful amounts of the nutrients,

either because they were iron replete, or because they did not consume sufficient amounts of

the supplement. This is why the intervention did not show a significant impact on participants’

Hb levels.

4.2 LIMITATIONS

The intervention only took place during school days but not on school holidays, weekends or

public holidays. The study was also limited to 14 weeks instead of the envisaged 16 weeks

because of a strike action mounted by teachers in the public schools. The unforeseen break

may have been partly responsible for the insignificant effect of the intervention on learner’s Hb

levels. Research ethics imposed another limiting factor in that its disapproval of participation by

children with a very low iron status naturally precluded participation by moderately and

severely anemic children. It is understandable, therefore, that absorption in children whose

iron status was replete was less pronounced than it could have been with moderately or

severely anemic children. The lack of dietary data was also a limitation as it would have helped

in the interpretation of the intervention results,

A further limitation that may have affected the outcome of the study was the fact that the

product was not subjected to a consumer sensory evaluation test before it was administered,

with the result that no data were obtained about its acceptability prior to the intervention. It

stands to reason, therefore, that a responsive flavour adjustment to appease participants’

preference might have proved instrumental in increasing consumption, which could have led to

different results.

58

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS

It can be concluded from measurements of Hb levels that the maize-based liquid meal

supplement enriched with multiple micronutrients administered to participants in the study at

issue did not have a significant effect on their iron status, possibly owing to a low prevalence of

anemia and, for that reason, a low rate of consumption and therefore of iron absorption among

members of the participating group.

It can also be concluded from anthropometric data that the liquid meal supplement did not

have a significant effect on the nutritional status of participants.

RECOMMENDATIONS

It is recommended that the intervention be taken to a primary school where the

children are really needy and anemic to see if a significant effect will be forthcoming

from the intervention.

Since the poor consumption observed in the study under review could have been a

result of aversion to the taste (vanilla flavor) of the maize-based liquid meal supplement

enriched with multiple micronutrients, it is recommended that the chocolate flavoured

product be substituted, since it proved to be the most liked in the consumer sensory

evaluation which was carried out later.

To increase consumption children should be given the supplement between meals when

the digestive process is sufficiently advanced to prevent interference with absorption of

the iron supplement.

It is also recommended that the female participants be questioned in retrospect to

discover whether any of them had begun to menstruate at the time of experimental

supplementation, and that a statistical analysis be done accordingly to accommodate

the outcome of what the recommended questioning reveals.

59

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74

ADDENDUM 1: ETHICAL APPROVAL

75

ADDENDUM 2: ASSENT FORM FOR 7-8 YEARS FOR CLINICAL TRIAL/INTERVENTION RESEARCH

ASSENT FORM FOR PROTOCOL TITLE: Effect of enriched maize-based liquid meal supplement

on the cognitive performance of primary school children.

We wish to know if you would like to volunteer to be part of a research study in which you will

consume a maize – based- ready- to- use meal supplement (let’s call it a maize sip for short).

We are asking you to help us to gather information on the effect of this on learning and

concentration abilities (cognitive performance) and the amount of iron in the bodies of

children. It will also tell us if it improves nutritional status in children like you.

About 160 children are going to take part in this study, and the study will last for 16 weeks over

the first two school terms of the year. During that time you will have to consume one of two

maize sips 5 days a week for the 16 week study period at a specific location at the school. The

sip you will receive will be determined by chance like flipping a coin.

During the study you will undergo different kinds of procedures and tests. We will measure

your weight and height and assess your learning and concentration abilities (cognitive

performance). This will be done before and after the study period. At the beginning and end of

the study, they will also take a tiny amount of blood (5 drops) from your finger. This may hurt,

but will only take a minute. These tests will take about 3 hours in total but will only take place

two times during the whole study.

You will receive deworming medication, 1 tablet, once off before the start of the study. The

unpleasant effects that can occur after taking the medication may include: temporary stomach

pain, diarrhea and vomiting, breaking out with a rash and hives, headaches and agranulocytosis

(sudden fever, shaking and sore throat).We will give you a sickness diary in which you or your

parents must record every time you are ill. It is very important that you tell your doctor, nurse

or your parents if you don’t feel well at anytime during the study.

If you do not want to take part anymore you may decide at anytime during the study to stop

participating, no one will force you to carry on. No one will be cross or upset with you if you

don’t want to. You don’t have to give us your answer now, take your time and read through the

form again before you decide. If you sign at the bottom it will mean that you have read this

paper and that you would like to be in this study.

76

Your name Person obtaining consent Parent /Guardian as

witness

Name

Signature

Date

15 July 2010

77

ADDENDUM 3:

NAME BADGES FOR CHILDREN

78

ADDENDUM 4: COMPLIANCE SHEET

COMPLIANCE JULY – NOV 2010 NAME:……………………………………… ………………………………………………… SUBJECT #: GRADE:………………..

Monday Tuesday Wednesday Thursday Friday COMMENTS

WK1 July 19 20 21 22 23

…………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………..……………………………………………………………………………………………………………………………

WK2 26 27 28 29 30

WK3 2 3 4 5 6

WK4 9 10 11 12 13

Public holiday

WK5 16 17 18 19 20

WK6 23 24 25 26 27

WK7 30 31 Sept. 1 2 3

WK8 6 7 8 9 10

WK9 13 14 15 16 17

WK10 20 21 22 23 24

Schoolholiday

27 28 29 30 Oct. 1

SCHOOL HOLIDAY

WK11 4 5 6 7 8

WK12 11 12 13 14 15

WK13 18 19 20 21 22

WK14 25 26 27 28 29

WK15 Nov. 1 2 3 4 5

WK16 8 9 10 11 12

79

ADDENDUM 5: SOCIO-DEMOGRAPHIC QUESTIONNAIRE Subject no.

Birth date:

Interview date: Child’s name……………………………………………………………………………Gender: M F Address …………………………………………………………………………………Religion………………………………………… ………………………………………………………………………………………………..Mother’s language………………………. Tel :(H)…………………………………………………………………………(W)…………………………………………………………… 1. Relationship to child: Mother Father Grandparent Sibling Aunt/Uncle Other 2.Household composition

Name of household members

Age (yrs)

Gender Family Relationship to the child

Does this person eat and sleep at home at least 4 days a week?

M F Relationship Code Yes No

Relationship (use as child reference): Father (1), Mother (2),Sibling (3),Grandmother (4), Grandfather(5), Aunt (6). Uncle (7), Cousin (8), Friend (9), Other (10)

3. Marital status of mother (tick one)

1 2 3 4 5 6 7 8

Unmarried Married Divorced Separated Widowed Living together

Traditional marriage

Other please specify

Tick one block only for every question

fath

er

Mo

ther

Sib

ling

gran

dm

a

Gra

nd

pa

un

cle

Au

nt

cou

sin

frie

nd

Oth

er

4. Who is mainly responsible for food preparation in the house

1 2 3 4 5 6 7 8 9 10

5. Who decides on what type of foods are bought for the household?

1 2 3 4 5 6 7 8 9 10

6. Who is mainly responsible for serving or feeding the child

1 2 3 4 5 6 7 8 9 10

7. Who is the head of this household?

1 2 3 4 5 6 7 8 9 10

8. Who decides how much is 1 2 3 4 5 6 7 8 9 10

80

Now look at this child and tick one block of every question

9. Would you consider this to be a healthy child?

1 ………………………

2 ……………………

If no, specify

10. Is this child disabled? 1 ………………………..

2 ………………….

If yes, specify

Now decide on the following (considering where the child lives)

11. Type of dwelling: You can tick more than one block if necessary

1 Brick concrete

2 Traditional Mud

3 Tin

4 Plank, wood

5 Other Specify

12. number of people sleeping in the house for at least 4 nights per week?

13. Number of rooms in the house (excluding bathroom, toilet and kitchen, if separate):

14. Number of people per dwelling, living/sleeping (tick one)

1 0-2 persons

2 3-4 persons

3 More than 4 persons

15. Where do you get drinking water from? (tick one)

1 own tap

2 Communal tap

3 River, dam

4 Borehole Well

5 Other specify

16. What type of toilet does this household have? (tick one)

1 Flush

2 pit

3 bucket/ Pot

4 VIP

5 Other specify

17. What fuel is used for cooking most of the time? (you can tick more than one)

1 electric

2 gas

3 paraffin

4 wood

5 sun

6 Open fire

Tick one box only:

18. Does the child’s home have a working (i) Refrigerator/freezer

1 Fridge

2 Freezer

3 Both

4 None

(ii) Stove 1 Yes

2 No

If yes choose one ………………………. Gas, coal, electric

If yes choose one ………………….. With oven without oven

(iii) Primus or paraffin stove 1 Yes

2 No

(iv) Microwave 1 Yes

2 No

(v) Hot plate 1 Yes

2 No

(vi) Radio or Television 1 Radio

2 TV

3 Both

4 None

Now ask questions about:

spent on food?

81

19. Education level of mother (tick only one)

1 None

2 Primary school

3 Std 6 -8

4 Std 9 – 10

5 Tertiary education

6 Don’t know

20. Mother’s employment status (choose one)

1 House wife by choice

2 Unemployed

3 Self- employed

4 Wage earner

5 Other specify

6 Don’t know

21. Education level of caregiver (Tick only one)

1 None

2 Primary school

3 Std 6 -8

4 Std 9 – 10

5 Tertiary education

6 Not applicable

22. Father’s employment status (can tick more than one)

1 Unemployed

2 self employed

3 wage earner

4 retired by choice

5 other specify

6 not applicable e.g. deceased

23. How many people contribute to the total income (Tick one only)

1 1 person

2 2 persons

3 3-4 persons

4 5-6 persons

5 Over 6 persons

24. Household income per month (including wages ,rent, sales of veg. etc, state grants (Tick only one)

1 None

2 R100- R500

3 R500- R1000

4 R1000-R3000

5 R3000-R5000

6 Over R5000

7 Don’t know

25. Is this the usual income of the household? (Tick one box only)

1

2 If NO what other income is available? Specify

26. Is this more or less the income you had over the past six months? (Tick one only)

1 Yes

2 No

27. How much money is spent on food weekly? (Tick one only)

1 R0-R50

2 R50-R100

3 R100-R150

4 R150-R200

5 R200-R250

6 R250-R300

7 R300R350

8 R350-R400

9 Over R400

10 Don’t know

10 Don’t know

82

ADDENDUM 6

SICKNESS DIARY

NAME: _______________________ _________ SUBJECT #:______________

Condition Medication

Start

date

End

date

Name Dosage &

frequency

Start date End date

E.g. Cough 6 Feb

2010

8 Feb

2010

BRONCLEER 5ml

3 times a day

6 Feb

2010

8 Feb

2010

83